Specific antibody-drug-conjugates (adcs) with ksp inhibitors and anti-cd123-antibodies

ABSTRACT

The invention relates to specific Antibody-Drug-Conjugates (ADCs) with KSP inhibitors and anti-CD123-antibodies, to the use of these conjugates for the treatment and/or prophylaxis of diseases and to the use of these conjugates for preparing medicaments for treatment and/or prevention of diseases, in particular hyperproliferative and/or angiogenic disorders such as, for example, cancer diseases. Such treatments can be carried out as monotherapy or else in combination with other medicaments or further therapeutic measures.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.17/225,947, filed on Apr. 8, 2021, which is a continuation of U.S.application Ser. No. 16/310,285, filed on Dec. 14, 2018, which is theU.S. National Stage Entry of International Application No.PCT/EP2017/063951, filed internationally on Jun. 8, 2017, which claimsthe benefit of European Application No. 16174654.0, filed on Jun. 15,2016, each of which is incorporated herein by reference in its entirety.

SUBMISSION OF SEQUENCE LISTING

The instant application contains a Sequence Listing which has beensubmitted electronically in XML format and is hereby incorporated byreference in its entirety. Said XML copy, created on Jun. 15, 2023, isnamed 59362-717.303.xml and is 295,024 bytes in size.

INTRODUCTION AND STATE OF THE ART

The invention relates to specific Antibody-Drug-Conjugates (ADCs) withKSP (kinesin spindle protein) inhibitors and anti-CD123-antibodies, tothe use of these conjugates for the treatment and/or prophylaxis ofdiseases and to the use of these conjugates for preparing medicamentsfor treatment and/or prevention of diseases, in particularhyperproliferative and/or angiogenic disorders such as, for example,cancer diseases. Such treatments can be carried out as monotherapy orelse in combination with other medicaments or further therapeuticmeasures.

Cancer diseases are the consequence of uncontrolled cell growth of themost diverse tissues. In many cases, the new cells penetrate intoexisting tissue (invasive growth), or they metastase into remote organs.Cancer diseases occur in the most diverse organs and often havetissue-specific courses of the disease. The term cancer as a genericterm therefore describes a large group of defined diseases of variousorgans, tissue and cell types.

Tumours in early stages can possibly be removed by surgical andradiotherapy measures. Metastased tumours as a rule can only be treatedpalliatively by chemotherapeutics. The aim here is to achieve theoptimum combination of an improvement in the quality of life andprolonging of life.

Conjugates of binder proteins and antibodies with one or more activecompound molecules are known, in particular in the form of antibody drugconjugates (ADCs) in which an internalising antibody directed against atumour-associated antigen is covalently attached via a linker to acytotoxic agent. Following introduction of the ADCs into the tumour celland subsequent dissociation of the conjugate, either the cytotoxic agentitself or a cytotoxic metabolite formed therefrom is released within thetumour cell and can unfold its action therein directly and selectively.In this manner, in contrast to conventional chemotherapy, damage tonormal tissue is contained in significantly narrower limits [see, forexample, J. M. Lambert, Curr. Opin. Pharmacol. 5, 543-549 (2005); A. M.Wu and P. D. Senter, Nat. Biotechnol. 23, 1137-1146 (2005); P. D.Senter, Curr. Opin. Chem. Biol. 13, 235-244 (2009); L. Ducry and B.Stump, Bioconjugate Chem. 21, 5-13 (2010)]. Thus, WO2012/171020describes ADCs in which a plurality of toxophor molecules are attachedvia a polymeric linker to an antibody. As possible toxophors,WO2012/171020 mentions, among others, the substances SB 743921, SB715992 (Ispinesib), MK-0371, AZD8477, AZ3146 and ARRY-520.

The substances mentioned last are kinesin spindle protein inhibitors.Kinesin spindle protein (KSP, also known as Eg5, HsEg5, KNSL1 or KIF11)is a kinesin-like motorprotein which is essential for the bipolarmitotic spindle to function. Inhibition of KSP leads to mitotic arrestand, over a relatively long term, to apoptosis (Tao et al., Cancer Cell2005 Jul. 8(1), 39-59). After the discovery of the firstcell-penetrating KSP inhibitor, Monastrol, KSP inhibitors haveestablished themselves as a class of novel chemotherapeutics (Mayer etal., Science 286: 971-974, 1999), and they are subject of a number ofpatent applications (e.g. WO2006/044825; WO2006/002236; WO2005/051922;WO2006/060737; WO03/060064; WO03/040979; and WO03/049527). However,since KSP unfolds its action only during a relatively short period oftime during the mitosis phase, KSP inhibitors have to be present in asufficiently high concentration during these initial phases.

In WO2015/096982 and WO2014/151030 antibody drug conjugates (ADCs) aredescribed with kinesin spindel Protein (KSP) inhibitors.

CD123 is the alpha chain of the IL-3 receptor and is also referred to asIL3R-alpha. CD123 is known to be expressed on primary AML samples andhas been reported on a number of malignant cells. Preferred anti-CD123antibodies are derived from antibodies disclosed by Sun et al. (Sun etal., 1996, Blood 87(1):83-92) and by Kuo et al. (Kuo et al., 2009,Bioconjug Chem. 20(10):1975-82).

Even with regard to the several already known antibody-drug-conjugates,there is still a high demand for new compounds that increase theavailability of active compounds that show acceptable or even betterproperties in comparison to the state of the art compounds.

SUMMARY OF THE INVENTION

Against this background it is an object of the present invention toprovide substances which, after administration at a relatively lowconcentration, unfold apoptotic action and may therefore be of benefitfor cancer therapy.

It has now surprisingly been found that antibody-drug-conjugates thathave a selected drug moiety as herein described together with a selectedantibody as herein described show acceptable and significant betterproperties.

Particular preference is given here to the extracellular cancer targetmolecule CD123 (IL-3Rα). This extracellular cancer target molecule isalso known as IL3RA (interleukin 3 receptor subunit alpha) and has theNCBI Gene ID: 3563.

For the target molecule CD123 antibody-drug-conjugates which provide avery potent and long lasting in vivo anti tumor efficacy have beenfound. Further antibody-drug-conjugates directed towards CD123 whichinduce a long-lasting tumor inhibition as indicated by the time perioduntil re-growth of the tumor occurs have been found.

To achieve this object, the invention further provides humanizedanti-CD123 antibodies which are in addition germlined, which means thesehave a very small deviation compared to antibody sequences of the humangermline. Special preference is given for the antibodies called“TPP-8987”, “TPP-9476”, “TPP-8988” and “TPP-9342”.

It has now surprisingly been found that a conjugate of an antibody (AK)or a functional fragment thereof together with one or more chemicalcompound(s) as defined in following formula (I) shows superiority overthe known conjugates:

-   -   in which    -   X is —CH₂— or —CH₂—CH₂—,    -   L is (—CH₂—CH₂—O—)_(m)—CH₂—CH₂—,    -   m is 1 to 20,    -   n is 1 to 8 and    -   AK is an anti-CD123 antibody or a functional fragment thereof        where the antibody or the functional fragment thereof is        attached via a cysteine residue and said antibody or functional        fragment thereof comprises a variable heavy chain comprising the        variable CDR1 sequence of the heavy chain, as shown in SEQ ID        NO: 122, the variable CDR2 sequence of the heavy chain, as shown        in SEQ ID NO: 123, and the variable CDR3 sequence of the heavy        chain, as shown in SEQ ID NO: 124, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 126, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            127, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 128, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 202, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            203, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 204, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 206, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            207, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 208, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 132, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            133, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 134, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 136, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            137, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 138, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 192, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            193, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 194, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 196, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            197, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 198,    -   and the salts, solvates, salts of the solvates and epimers        thereof.

DESCRIPTION OF THE FIGURES

FIG. 1 : Sequence protocol.

FIG. 2 : Annotated sequence of antibodies. For each of the antibodies orantibody fragments, the CDR regions (HCDR1, HCDR2, HCDR3, LCDR1, LCDR2,LCDR3) and the variable regions (VH, VL) are emphasized.

DETAILED DESCRIPTION OF THE INVENTION AND PARTICULAR EMBODIMENTS

Particularly preferred compounds of general formula (I) are those inwhich

-   -   X is —CH₂—,    -   L is (—CH₂—CH₂—O—)_(m)CH₂—CH₂—,    -   m is 1 to 20,    -   n is 1 to 8 and    -   AK is an anti-CD123 antibody or a functional fragment thereof        where the antibody or the functional fragment thereof is        attached via a cysteine residue and said antibody or functional        fragment thereof comprises        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 122, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            123, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 124, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 126, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            127, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 128, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 202, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            203, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 204, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 206, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            207, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 208, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 132, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            133, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 134, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 136, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            137, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 138, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 192, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            193, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 194, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 196, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            197, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 198,    -   and the salts, solvates, salts of the solvates and epimers        thereof.

Particularly preferred compounds of general formula (I) are those inwhich

-   -   X is —CH₂—,    -   L is (—CH₂—CH₂—O—)_(m)CH₂—CH₂—,    -   m is 1 to 20,    -   n is 1 to 8 and    -   AK is the anti-CD123 antibody TPP-8987, TPP-9476, TPP-8988, or        TPP-9342 or a functional fragment thereof where the antibody or        the functional fragment thereof is attached via a cysteine        residue,    -   and the salts, solvates, salts of the solvates and epimers        thereof.

More particularly preferred compounds of general formula (I) are thosein which

-   -   X is —CH₂—,    -   L is (—CH₂—CH₂—O—)_(m)CH₂—CH₂—,    -   m is 2 to 8,    -   n is 1 to 8 and    -   AK is an anti-CD123 antibody or a functional fragment thereof        where the antibody or the functional fragment thereof is        attached via a cysteine residue and said antibody or functional        fragment thereof comprises        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 122, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            123, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 124, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 126, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            127, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 128, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 202, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            203, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 204, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 206, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            207, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 208, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 132, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            133, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 134, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 136, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            137, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 138, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 192, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            193, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 194, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 196, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            197, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 198,    -   and the salts, solvates, salts of the solvates and epimers        thereof.

More particularly preferred compounds of general formula (I) are thosein which

-   -   X is —CH₂—,    -   L is (—CH₂—CH₂—O—)_(m)CH₂—CH₂—,    -   m is 2 to 8,    -   n is 1 to 8 and    -   AK is the anti-CD123 antibody TPP-8987, TPP-9476, TPP-8988, or        TPP-9342 or a functional fragment thereof where the antibody or        the functional fragment thereof is attached via a cysteine    -   and the salts, solvates, salts of the solvates and epimers        thereof.

More particularly preferred compounds of general formula (I) are:

-   -   and in which    -   n is 1 to 8 and    -   AK is an anti-CD123 antibody or a functional fragment thereof        where the antibody or the functional fragment thereof is        attached via a cysteine residue and said antibody or functional        fragment thereof comprises        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 122, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            123, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 124, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 126, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            127, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 128, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 202, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            203, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 204, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 206, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            207, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 208, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 132, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            133, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 134, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 136, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            137, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 138, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 192, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            193, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 194, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 196, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            197, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 198.

The most preferred compound of general formula (I) is:

-   -   and in which    -   n is 1 to 8 and    -   AK is the anti-CD123 antibody TPP-8987, TPP-9476, TPP-8988, or        TPP-9342 or a functional fragment thereof where the antibody or        the functional fragment thereof is attached via a cysteine        residue.

Further Description for Anti-CD123 Antibodies

The literature discloses various options of covalent coupling(conjugation) of organic molecules to antibodies. Preference accordingto the invention is given to the conjugation of the toxophores to theantibody via one or more sulphur atoms of cysteine residues of theantibody and/or via one or more NH groups of lysine residues of theantibody. However, it is also possible to bind the toxophor to theantibody via free carboxyl groups or via sugar residues of the antibody.

The antibody can be attached to the linker via a bond. Attachment of theantibody can be via a heteroatom of the binder. Heteroatoms according tothe invention of the antibody which can be used for attachment aresulphur (in one embodiment via a sulphhydryl group of the antibody),oxygen (according to the invention by means of a carboxyl or hydroxylgroup of the antibody) and nitrogen (in one embodiment via a primary orsecondary amine group or amide group of the antibody). These heteroatomsmay be present in the natural antibody or are introduced by chemicalmethods or methods of molecular biology. According to the invention, theattachment of the antibody to the toxophor has only a minor effect onthe binding activity of the antibody with respect to the targetmolecule. In a preferred embodiment, the attachment has no effect on thebinding activity of the antibody with respect to the target molecule.

In accordance with the present invention, the term “antibody” is to beunderstood in its broadest meaning and comprises immunoglobulinmolecules, for example intact or modified monoclonal antibodies,polyclonal antibodies or multispecific antibodies (e.g. bispecificantibodies). An immunoglobulin molecule preferably comprises a moleculehaving four polypeptide chains, two heavy chains (H chains) and twolight chains (L chains) which are typically linked by disulphidebridges. Each heavy chain comprises a variable domain of the heavy chain(abbreviated VH) and a constant domain of the heavy chain. The constantdomain of the heavy chain may, for example, comprise three domains CH1,CH2 and CH3. Each light chain comprises a variable domain (abbreviatedVL) and a constant domain. The constant domain of the light chaincomprises a domain (abbreviated CL). The VH and VL domains may besubdivided further into regions having hypervariability, also referredto as complementarity determining regions (abbreviated CDR) and regionshaving low sequence variability (framework region, abbreviated FR).Typically, each VH and VL region is composed of three CDRs and up tofour FRs. For example from the amino terminus to the carboxy terminus inthe following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. An antibodymay be obtained from any suitable species, e.g. rabbit, llama, camel,mouse or rat. In one embodiment, the antibody is of human or murineorigin. An antibody may, for example, be human, humanized or chimeric.

The term “monoclonal” antibody refers to antibodies obtained from apopulation of substantially homogeneous antibodies, i.e. individualantibodies of the population are identical except for naturallyoccurring mutations, of which there may be a small number. Monoclonalantibodies recognize a single antigenic binding site with highspecificity. The term monoclonal antibody does not refer to a particularpreparation process.

The term “intact” antibody refers to antibodies comprising both anantigen-binding domain and the constant domain of the light and heavychain. The constant domain may be a naturally occurring domain or avariant thereof having a number of modified amino acid positions.

The term “modified intact” antibody refers to intact antibodies fusedvia their amino terminus or carboxy terminus by means of a covalent bond(e.g. a peptide bond) with a further polypeptide or protein notoriginating from an antibody. Furthermore, antibodies may be modifiedsuch that, at defined positions, reactive cysteines are introduced tofacilitate coupling to a toxophor (see Junutula et al. Nat Biotechnol.2008, 26(8):925-32).

The term “human” antibody refers to antibodies which can be obtainedfrom a human or which are synthetic human antibodies. A “synthetic”human antibody is an antibody which is partially or entirely obtainablein silico from synthetic sequences based on the analysis of humanantibody sequences. A human antibody can be encoded, for example, by anucleic acid isolated from a library of antibody sequences of humanorigin. An example of such an antibody can be found in Soderlind et al.,Nature Biotech. 2000, 18:853-856.

The term “humanized” or “chimeric” antibody describes antibodiesconsisting of a non-human and a human portion of the sequence. In theseantibodies, part of the sequences of the human immunoglobulin(recipient) is replaced by sequence portions of a non-humanimmunoglobulin (donor). In many cases, the donor is a murineimmunoglobulin. In the case of humanized antibodies, amino acids of theCDR of the recipient are replaced by amino acids of the donor.Sometimes, amino acids of the framework, too, are replaced bycorresponding amino acids of the donor. In some cases the humanizedantibody contains amino acids present neither in the recepient nor inthe donor, which were introduced during the optimization of theantibody. In the case of chimeric antibodies, the variable domains ofthe donor immunoglobulin are fused with the constant regions of a humanantibody.

The term complementarity determining region (CDR) as used herein refersto those amino acids of a variable antibody domain which are requiredfor binding to the antigen. Typically, each variable region has threeCDR regions referred to as CDR1, CDR2 and CDR3. Each CDR region mayembrace amino acids according to the definition of Kabat and/or aminoacids of a hypervariable loop defined according to Chotia. Thedefinition according to Kabat comprises, for example, the region fromabout amino acid position 24-34 (CDR1), 50-56 (CDR2) and 89-97 (CDR3) ofthe variable light chain and 31-35 (CDR1), 50-65 (CDR2) and 95-102(CDR3) of the variable heavy chain (Kabat et al., Sequences of Proteinsof Immunological Interest, 5th Ed. Public Health Service, NationalInstitutes of Health, Bethesda, MD. (1991)). The definition according toChotia comprises, for example, the region from about amino acid position26-32 (CDR1), 50-52 (CDR2) and 91-96 (CDR3) of the variable light chainand 26-32 (CDR1), 53-55 (CDR2) and 96-101 (CDR3) of the variable heavychain (Chothia and Lesk; J Mol Biol 196: 901-917 (1987)). In some cases,a CDR may comprise amino acids from a CDR region defined according toKabat and Chotia.

Depending on the amino acid sequence of the constant domain of the heavychain, antibodies may be categorized into different classes. There arefive main classes of intact antibodies: IgA, IgD, IgE, IgG and IgM, andseveral of these can be divided into further subclasses. (Isotypes),e.g. IgG1, IgG2, IgG3, IgG4, IgA1 and IgA2. The constant domains of theheavy chain, which correspond to the different classes, are referred toas [alpha/a], [delta/6], [epsilon/c], [gamma/y] and [my/1.1]. Both thethree-dimensional structure and the subunit structure of antibodies areknown.

The term “functional fragment” or “antigen-binding antibody fragment” ofan antibody/immunoglobulin is defined as a fragment of anantibody/immunoglobulin (e.g. the variable domains of an IgG) whichstill comprises the antigen binding domains of theantibody/immunoglobulin. The “antigen binding domain” of an antibodytypically comprises one or more hypervariable regions of an antibody,for example the CDR, CDR2 and/or CDR3 region. However, the “framework”or “skeleton” region of an antibody may also play a role during bindingof the antibody to the antigen. The framework region forms the skeletonof the CDRs. Preferably, the antigen binding domain comprises at leastamino acids 4 to 103 of the variable light chain and amino acids 5 to109 of the variable heavy chain, more preferably amino acids 3 to 107 ofthe variable light chain and 4 to 111 of the variable heavy chain,particularly preferably the complete variable light and heavy chains,i.e. amino acids 1-109 of the VL and 1 to 113 of the VH (numberingaccording to WO97/08320).

“Functional fragments” or “antigen-binding antibody fragments” of theinvention encompass, non-conclusively, Fab, Fab′, F(ab′)2 and Fvfragments, diabodies, Single Domain Antibodies (DAbs), linearantibodies, individual chains of antibodies (single-chain Fv,abbreviated to scFv); and multispecific antibodies, such as bi andtri-specific antibodies, for example, formed from antibody fragments C.A. K Borrebaeck, editor (1995) Antibody Engineering (Breakthroughs inMolecular Biology), Oxford University Press; R. Kontermann & S. Duebel,editors (2001) Antibody Engineering (Springer Laboratory Manual),Springer Verlag. Antibodies other than “multispecific” or“multifunctional” antibodies are those having identical binding sites.Multispecific antibodies may be specific for different epitopes of anantigen or may be specific for epitopes of more than one antigen (see,for example, WO 93/17715; WO 92/08802; WO 91/00360; WO 92/05793; Tutt,et al., 1991, J. Immunol. 147:60 69; U. S. Pat. Nos. 4,474,893;4,714,681; 4,925,648; 5,573,920; 5,601,819; or Kostelny et al., 1992, J.Immunol. 148: 1547 1553). An F(ab′)₂ or Fab molecule may be constructedsuch that the number of intermolecular disulphide interactions occurringbetween the Ch1 and the CL domains can be reduced or else completelyprevented.

“Epitopes” refer to protein determinants capable of binding specificallyto an immunoglobulin or T cell receptors. Epitopic determinants usuallyconsist of chemically active surface groups of molecules such as aminoacids or sugar side chains or combinations thereof, and usually havespecific 3-dimensional structural properties and also specific chargeproperties.

“Functional fragments” or “antigen-binding antibody fragments” may befused with another polypeptide or protein, not originating from anantibody, via the amino terminus or carboxyl terminus thereof, by meansof a covalent bond (e.g. a peptide linkage). Furthermore, antibodies andantigen-binding fragments may be modified by introducing reactivecysteines at defined locations, in order to facilitate coupling to atoxophore (see Junutula et al. Nat Biotechnol. 2008 August;26(8):925-32).

Polyclonal antibodies can be prepared by methods known to a person ofordinary skill in the art. Monoclonal antibodies may be prepared bymethods known to a person of ordinary skill in the art (Köhler andMilstein, Nature, 256, 495-497, 1975). Human and humanized monoclonalantibodies may be prepared by methods known to a person of ordinaryskill in the art (Olsson et al., Meth Enzymol. 92, 3-16 or Cabilly et alU.S. Pat. No. 4,816,567 or Boss et al U.S. Pat. No. 4,816,397).

A person of ordinary skill in the art is aware of diverse methods forpreparing human antibodies and fragments thereof, such as, for example,by means of transgenic mice (N Lonberg and D Huszar, Int Rev Immunol.1995; 13(1):65-93) or phage display technologies (Clackson et al.,Nature. 1991 Aug. 15; 352(6336):624-8). Antibodies of the invention maybe obtained from recombinant antibody libraries consisting for exampleof the amino acid sequences of a multiplicity of antibodies compiledfrom a large number of healthy volunteers. Antibodies may also beproduced by means of known recombinant DNA technologies. The nucleicacid sequence of an antibody can be obtained by routine sequencing or isavailable from publically accessible databases.

An “isolated” antibody or binder has been purified to remove otherconstituents of the cell. Contaminating constituents of a cell which mayinterfere with a diagnostic or therapeutic use are, for example,enzymes, hormones, or other peptidic or non-peptidic constituents of acell. A preferred antibody or binder is one which has been purified toan extent of more than 95% by weight, relative to the antibody or binder(determined for example by Lowry method, UV-Vis spectroscopy or by SDScapillary gel electrophoresis). Moreover an antibody which has beenpurified to such an extent that it is possible to determine at least 15amino acids of the amino terminus or of an internal amino acid sequence,or which has been purified to homogeneity, the homogeneity beingdetermined by SDS-PAGE under reducing or non-reducing conditions(detection may be determined by means of Coomassie Blue staining orpreferably by silver coloration). However, an antibody is normallyprepared by one or more purification steps.

The term “specific binding” or “binds specifically” refers to anantibody or binder which binds to a predetermined antigen/targetmolecule. Specific binding of an antibody or binder typically describesan antibody or binder having an affinity of at least 10⁻⁷ M (as Kdvalue; i.e. preferably those with Kd values smaller than 10⁻⁷ M), withthe antibody or binder having an at least two times higher affinity forthe predetermined antigen/target molecule than for a non-specificantigen/target molecule (e.g. bovine serum albumin, or casein) which isnot the predetermined antigen/target molecule or a closely relatedantigen/target molecule. The antibodies preferably have an affinity ofat least 10⁻⁷ M (as Kd value; in other words preferably those withsmaller Kd values than 10⁻⁷ M), preferably of at least 10⁻⁸ M, morepreferably in the range from 10⁻⁹ M to 10⁻¹¹ M. The Kd values may bedetermined, for example, by means of surface plasmon resonancespectroscopy.

The antibody-drug conjugates of the invention likewise exhibitaffinities in these ranges. The affinity is preferably not substantiallyaffected by the conjugation of the drugs (in general, the affinity isreduced by less than one order of magnitude, in other words, forexample, at most from 10⁻⁸ M to 10⁻⁷ M).

The antibodies used in accordance with the invention are also notablepreferably for a high selectivity. A high selectivity exists when theantibody of the invention exhibits an affinity for the target proteinwhich is better by a factor of at least 2, preferably by a factor of 5or more preferably by a factor of 10, than for an independent otherantigen, e.g. human serum albumin (the affinity may be determined, forexample, by means of surface plasmon resonance spectroscopy).

Furthermore, the antibodies of the invention that are used arepreferably cross-reactive. In order to be able to facilitate and betterinterpret preclinical studies, for example toxicological or activitystudies (e.g. in xenograft mice), it is advantageous if the antibodyused in accordance with the invention not only binds the human targetprotein but also binds the species target protein in the species usedfor the studies. In one embodiment the antibody used in accordance withthe invention, in addition to the human target protein, iscross-reactive to the target protein of at least one further species.For toxicological and activity studies it is preferred to use species ofthe families of rodents, dogs and non-human primates. Preferred rodentspecies are mouse and rat. Preferred non-human primates are rhesusmonkeys, chimpanzees and long-tailed macaques.

In one embodiment the antibody used in accordance with the invention, inaddition to the human target protein, is cross-reactive to the targetprotein of at least one further species selected from the group ofspecies consisting of mouse, rat and long-tailed macaque (Macacafascicularis). Especially preferred are antibodies used in accordancewith the invention which in addition to the human target protein are atleast cross-reactive to the mouse target protein. Preference is given tocross-reactive antibodies whose affinity for the target protein of thefurther non-human species differs by a factor of not more than 50, moreparticularly by a factor of not more than ten, from the affinity for thehuman target protein.

Antibodies Directed Against a Cancer Target Molecule

The target molecule towards which the binder, for example an antibody oran antigen-binding fragment thereof, is directed is preferably a cancertarget molecule. The term “cancer target molecule” describes a targetmolecule which is more abundantly present on one or more cancer cellspecies than on non-cancer cells of the same tissue type. Preferably,the cancer target molecule is selectively present on one or more cancercell species compared with non-cancer cells of the same tissue type,where selectively describes an at least two-fold enrichment on cancercells compared to non-cancer cells of the same tissue type (a “selectivecancer target molecule”). The use of cancer target molecules allows theselective therapy of cancer cells using the conjugates according to theinvention.

Particular preference is given here to the extracellular cancer targetmolecule IL-3Rα, CD123 (SEQ ID NO: 221). This extracellular cancertarget molecule is also known as IL3RA (interleukin 3 receptor subunitalpha) and has the NCBI Gene ID: 3563.

The functional interleukin 3 receptor is a heterodimer which comprisesthe specific alpha chain (IL-3Rα, CD123) and a “general” IL-3 receptorbeta chain ((βC, CD131) which is shared with the receptors for thegranolocyte macrophage colony stimulating factor (GM-CSF) andinterleukin 5 (IL-5).

IL-3Rα, CD123, is a transmembrane protein of type 1 having a calculatedmolecular weight of about 41 kDa. CD123 comprises an extracellulardomain involved in IL-3 binding, a transmembrane domain and a shortcytoplasmic end of about 50 amino acids. The extracellular domainconsists of two regions: an N-terminal region of about 100 amino acidshaving sequence similarity to the equivalent regions of the GM-CSF andthe IL-5 receptor alpha chain, and a region proximal to thetransmembrane domain which region comprises four conserved cysteineresidues and a WSXWS motif common in the cytokine receptor family.

The IL-3 binding domain comprises a cytokine receptor motif (CRM) ofabout 200 amino acid residues which is constructed of two domains whichare folded Ig-like. The extracellular domain of IL-3Rα, CD123, is highlyglycosylated, the N-glycosylation being required for ligand binding andreceptor signal transduction.

IL-3Rα, CD123, is expressed extensively throughout the haematopoieticsystem, for example on haematopoietic precursor cells, mast cells,erythroid cells, megakaryocytes, neutrophil, basophil and eosinophilgranulocytes, monocytes/macrophages, and CDS+B lymphocytes; CD123 isalso expressed on non-haematopoietic cells such as dendritic cells,Leydig cells, endothelial cells and stromal cells.

IL-3Rα, CD123, is also expressed by cells involved in certain diseases;these diseases comprise: myelodysplastic syndrome, leukaemia (such asacute myeloid leukaemia (AML)), lymphoma, allergies and autoimmunedisorders such as lupus or scleroderma.

Owing to these relationships, anti-IL-3Rα, anti-CD123, antibodies can beemployed in therapy as naked antibodies or coupled antibodies (such asADCs).

The present invention relates to compounds coupled to antibodiesspecifically binding to IL-3Rα, CD123 (SEQ ID NO: 221).

The term “anti-CD123 antibody” relates to an antibody which specificallybinds the cancer target molecule CD123 (IL-3Rα, SEQ ID NO: 221),preferentially with an affinity which is sufficient for a diagnosticand/or therapeutic application. In one embodiment, binding of ananti-CD123 antibody to a protein not related to CD123 is less than 10%of the binding of the antibody to CD123, determined, for example, bysurface plasmon resonance spectroscopy. In certain embodiments, theantibody binds CD123 (IL-3Rα, SEQ ID NO: 221) with a dissociationconstant (KD) of ≤1 μM≤100 nM, ≤10 nM, ≤1 nM, ≤0.1 nM, ≤0.01 nM, or≤0.001 nM. In certain embodiments, the anti-CD123 antibody binds to anepitope which is conserved between different species.

Antibodies which bind cancer target molecules may be prepared by aperson of ordinary skill in the art using known processes, such as, forexample, chemical synthesis or recombinant expression. Binders forcancer target molecules may be acquired commercially or may be preparedby a person of ordinary skill in the art using known processes, such as,for example, chemical synthesis or recombinant expression. Furtherprocesses for preparing antibodies or antigen-binding antibody fragmentsare described in WO 2007/070538 (see page 22 “Antibodies”). The personskilled in the art knows how processes such as phage display libraries(e.g. Morphosys HuCAL Gold) can be compiled and used for discoveringantibodies or antigen-binding antibody fragments (see WO 2007/070538,page 24 ff and AK Example 1 on page 70, AK Example 2 on page 72).Further processes for preparing antibodies that use DNA libraries from Bcells are described for example on page 26 (WO 2007/070538). Processesfor humanizing antibodies are described on page 30-32 of WO2007070538and in detail in Queen, et al., Pros. Natl. Acad. Sci. USA86:10029-10033,1989 or in WO 90/0786. Furthermore, processes for therecombinant expression of proteins in general and of antibodies inparticular are known to the person skilled in the art (see, for example,in Berger and Kimmel (Guide to Molecular Cloning Techniques, Methods inEnzymology, Vol. 152, Academic Press, Inc.); Sambrook, et al.,(Molecular Cloning: A Laboratory Manual, (Second Edition, Cold SpringHarbor Laboratory Press; Cold Spring Harbor, N.Y.; 1989) Vol. 1-3);Current Protocols in Molecular Biology, (F. M. Ausabel et al. [Eds.],Current Protocols, Green Publishing Associates, Inc./John Wiley & Sons,Inc.); Harlow et al., (Monoclonal Antibodies: A Laboratory Manual, ColdSpring Harbor Laboratory Press (19881, Paul [Ed.]); FundamentalImmunology, (Lippincott Williams & Wilkins (1998)); and Harlow, et al.,(Using Antibodies: A Laboratory Manual, Cold Spring Harbor LaboratoryPress (1998)). The person skilled in the art knows the correspondingvectors, promoters and signal peptides which are necessary for theexpression of a protein/antibody. Commonplace processes are alsodescribed in WO 2007/070538 on pages 41-45. Processes for preparing anIgG1 antibody are described for example in WO 2007/070538 in Example 6on page 74 ff. Processes which allow the determination of theinternalization of an antibody after binding to its antigen are known tothe skilled person and are described for example in WO 2007/070538 onpage 80. The person skilled in the art is able to use the processesdescribed in WO 2007/070538 that have been used for preparingcarboanhydrase IX (Mn) antibodies in analogy for the preparation ofantibodies with different target molecule specificity.

Anti-CD123 Antibodies

According to the invention, use is made of an anti-CD123 antibody or anantigen-binding fragment thereof, preferably one selected from thosedescribed below or modified by suitable mutation. In addition, theperson skilled in the art is familiar with antibodies binding to CD123.

Sun et al. (Sun et al., 1996, Blood 87(1):83-92) describe the generationand properties of the monoclonal antibody 7G3, which binds to theN-terminal domain of IL-3Rα, CD123. U.S. Pat. No. 6,177,078 (Lopez)relates to the anti-CD123 antibody 7G3. A chimeric variant of thisantibody (CSL360) is described in WO 2009/070844, and a humanizedversion (CSL362) in WO 2012/021934. The sequence of the 7G3 antibody isdisclosed in EP2426148.

This 7G3 sequence represents the starting point of the humanized andgermlined antibodies TPP-8987 and TPP-9476.

An antibody which, after cell surface antigen binding, is internalizedparticularly well is the anti-CD123 antibody 12F1 disclosed by Kuo etal. (Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82). The antibody12F1 binds with higher affinity to CD123 than the antibody 7G3 and,after cell surface antigen binding, is internalized markedly faster than7G3. Bispecific scFv immunofusion proteins based on 12F1 are disclosedin WO 2013/173820.

This 12F1 sequence represents the starting point of the humanized andgermlined antibodies TPP-8988 and TPP-9342.

The invention relates in particular to conjugates with antibodies orantigen-binding antibody fragments thereof or variants thereof derivedfrom the antibodies 7G3 (Sun et al., 1996, Blood 87(1):83-92) and 12F1(Kuo et al., 2009, Bioconjug Chem. 20(10):1975-82) originated from themouse, or to conjugates with antibodies or antigen-binding antibodyfragments thereof or variants thereof derived from the antibody 12F1(Kuo et al., 2009, Bioconjug Chem. 20(10): 1975-82) originating from themouse.

Generation of the Anti-CD123 Antibodies

Based on the publication of the sequences of the variable regions (VHand VL) of 7G3 (EP2426148), the following antibody sequences wereobtained by CDR grafting in human framework regions and subsequentgermlining optimization: TPP-8987 and TPP-9476.

Based on the publication of the sequences of the variable regions (VHand VL) of 12F1 (WO 2013/173820), the following antibody were obtainedby CDR grafting in human framework regions and subsequent germliningoptimization: TPP-8988 and TPP-9342.

Particular Embodiments of Anti-CD123 Antibodies

In the present application, reference is made to the following preferredanti-CD123 antibodies of the invention, as shown in the table below:“TPP-8987”, “TPP-9476”, “TPP-8988” and “TPP-9342”.

TPP-8987 and TPP-9476 are humanized and germlined variants of theantibody 7G3.

TPP-8988 and TPP-9342 are humanized and germlined variants of theantibody 12F1

As shown in the examples it was a further object of this invention toprovide antibodies of 7G3 and 12F1 which are humanized and which arevery close to the human antibody germline sequences (germlined).Therefore antibodies TPP-8987, TPP-9476, TPP-8988, and TPP-9342 arefurther embodiments of this invention. The sequences of these antibodiesare shown in the following table:

TABLE Table of preferred anti-CD123antibodies for ADCs: SEQ ID NO: HeavyLight Chain Chain Antibody VH H-CDR1 H-CDR2 H-CDR3 VL L-CDR1 L-CDR2L-CDR3 (IgG) (IgG) TPP-8987 121 122 123 124 125 126 127 128 129 130TPP-9476 201 202 203 204 205 206 207 208 209 210 TPP-8988 131 132 133134 135 136 137 138 139 140 TPP-9342 191 192 193 194 195 196 197 198 199200

Particular preference is given for ADCs to the anti-CD123 antibodiescalled “TPP-8987”, “TPP-9476”, “TPP-8988” and “TPP-9342”.

TPP-8987 is an anti-CD123 antibody which comprises a variable heavychain comprising the variable CDR1 sequence of the heavy chain, as shownin SEQ ID NO: 122, the variable CDR2 sequence of the heavy chain, asshown in SEQ ID NO: 123, and the variable CDR3 sequence of the heavychain, as shown in SEQ ID NO: 124, and a variable light chain comprisingthe variable CDR1 sequence of the light chain, as shown in SEQ ID NO:126, the variable CDR2 sequence of the light chain, as shown in SEQ IDNO: 127, and the variable CDR3 sequence of the light chain, as shown inSEQ ID NO: 128. TPP-9476 is an anti-CD123 antibody which comprises avariable heavy chain comprising the variable CDR1 sequence of the heavychain, as shown in SEQ ID NO: 202, the variable CDR2 sequence of theheavy chain, as shown in SEQ ID NO: 203, and the variable CDR3 sequenceof the heavy chain, as shown in SEQ ID NO: 204, and a variable lightchain comprising the variable CDR1 sequence of the light chain, as shownin SEQ ID NO: 206, the variable CDR2 sequence of the light chain, asshown in SEQ ID NO: 207, and the variable CDR3 sequence of the lightchain, as shown in SEQ ID NO: 208. TPP-8988 is an anti-CD123 antibodywhich comprises a variable heavy chain comprising the variable CDR1sequence of the heavy chain, as shown in SEQ ID NO: 132, the variableCDR2 sequence of the heavy chain, as shown in SEQ ID NO: 133, and thevariable CDR3 sequence of the heavy chain, as shown in SEQ ID NO: 134,and a variable light chain comprising the variable CDR1 sequence of thelight chain, as shown in SEQ ID NO: 136, the variable CDR2 sequence ofthe light chain, as shown in SEQ ID NO: 137, and the variable CDR3sequence of the light chain, as shown in SEQ ID NO: 138.

TPP-9342 is an anti-CD123 antibody which comprises a variable heavychain comprising the variable CDR1 sequence of the heavy chain, as shownin SEQ ID NO: 192, the variable CDR2 sequence of the heavy chain, asshown in SEQ ID NO: 193, and the variable CDR3 sequence of the heavychain, as shown in SEQ ID NO: 194, and a variable light chain comprisingthe variable CDR1 sequence of the light chain, as shown in SEQ ID NO:196, the variable CDR2 sequence of the light chain, as shown in SEQ IDNO: 197, and the variable CDR3 sequence of the light chain, as shown inSEQ ID NO: 198.

TPP-8987 is an anti-CD123 antibody which preferably comprises a variableregion of the heavy chain corresponding to SEQ ID NO: 121 and a variableregion of the light chain corresponding to SEQ ID NO: 125.

TPP-9476 is an anti-CD123 antibody which preferably comprises a variableregion of the heavy chain corresponding to SEQ ID NO: 201 and a variableregion of the light chain corresponding to SEQ ID NO: 205.

TPP-8988 is an anti-CD123 antibody which preferably comprises a variableregion of the heavy chain corresponding to SEQ ID NO: 131 and a variableregion of the light chain corresponding to SEQ ID NO: 135.

TPP-9342 is an anti-CD123 antibody which preferably comprises a variableregion of the heavy chain corresponding to SEQ ID NO: 191 and a variableregion of the light chain corresponding to SEQ ID NO: 195.

TPP-8987 is an anti-CD123 antibody preferably comprising a region of theheavy chain corresponding to SEQ ID NO: 129 and a region of the lightchain corresponding to SEQ ID NO: 130.

TPP-9476 is an anti-CD123 antibody preferably comprising a region of theheavy chain corresponding to SEQ ID NO: 209 and a region of the lightchain corresponding to SEQ ID NO: 210.

TPP-8988 is an anti-CD123 antibody comprising a region of the heavychain corresponding to SEQ ID NO: 139 and a region of the light chainpreferably corresponding to SEQ ID NO: 140. TPP-9342 is an anti-CD123antibody preferably comprising a region of the heavy chain correspondingto SEQ ID NO: 199 and a region of the light chain corresponding to SEQID NO: 200.

Preferred embodiments of the anti-CD123 antibody for coupling withlinkers and/or toxophores according to the invention are those below:

-   -   1. An antibody or an antigen-binding fragment thereof binding to        CD123, comprising:        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 122, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            123, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 124, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 126, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            127, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 128, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 202, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            203, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 204, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 206, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            207, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 208, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 132, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            133, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 134, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 136, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            137, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 138, or        -   a variable heavy chain comprising the variable CDR1 sequence            of the heavy chain, as shown in SEQ ID NO: 192, the variable            CDR2 sequence of the heavy chain, as shown in SEQ ID NO:            193, and the variable CDR3 sequence of the heavy chain, as            shown in SEQ ID NO: 194, and        -   a variable light chain comprising the variable CDR1 sequence            of the light chain, as shown in SEQ ID NO: 196, the variable            CDR2 sequence of the light chain, as shown in SEQ ID NO:            197, and the variable CDR3 sequence of the light chain, as            shown in SEQ ID NO: 198.    -   2. The antibody or an antigen-binding fragment thereof according        to embodiment 1, comprising:        -   a variable sequence of the heavy chain, as shown in SEQ ID            NO:121, and a variable sequence of the light chain, as shown            in SEQ ID NO:125, or        -   a variable sequence of the heavy chain, as shown in SEQ ID            NO:201, and a variable sequence of the light chain, as shown            in SEQ ID NO:205, or        -   a variable sequence of the heavy chain, as shown in SEQ ID            NO:131, and a variable sequence of the light chain, as shown            in SEQ ID NO:135, or        -   a variable sequence of the heavy chain, as shown in SEQ ID            NO:191, and a variable sequence of the light chain, as shown            in SEQ ID NO:195.    -   3. The antibody according to any of the preceding embodiments        which is an IgG antibody.    -   4. The antibody according to any of the preceding embodiments,        comprising:        -   a sequence of the heavy chain, as shown in SEQ ID NO:129,            and a sequence of the light chain, as shown in SEQ ID            NO:130, or        -   a sequence of the heavy chain, as shown in SEQ ID NO:209,            and a sequence of the light chain, as shown in SEQ ID            NO:210, or        -   a sequence of the heavy chain, as shown in SEQ ID NO:139,            and a sequence of the light chain, as shown in SEQ ID            NO:140, or        -   a sequence of the heavy chain, as shown in SEQ ID NO:199,            and a sequence of the light chain, as shown in SEQ ID            NO:200.    -   5. The antibody according to any of the preceding embodiments,        comprising: The antigen-binding fragment according to any of the        preceding embodiments or an antigen-binding fragment of an        antibody according to any of the preceding embodiments which is        an scFv, Fab, Fab′ fragment or a F(ab)2 fragment.    -   6. The antibody or the antigen-binding fragment according to any        of the preceding embodiments which is a monoclonal antibody or        an antigen-binding fragment thereof    -   7. The antibody or the antigen-binding fragment according to any        of the preceding embodiments which is a human, humanized or        chimeric antibody or an antigen-binding fragment.    -   Particular preference is given to the anti-CD123 antibodies        called “TPP-8987”, “TPP-9476”, “TPP-8988” and “TPP-9342”.

DNA Molecules of the Invention

The present invention also relates to the DNA molecules that encode anantibody of the invention or antigen-binding fragment thereof. Thesesequences are optimized in certain cases for mammalian expression. DNAmolecules of the invention are not limited to the sequences disclosedherein, but also include variants thereof. DNA variants within theinvention may be described by reference to their physical properties inhybridization. The skilled worker will recognize that DNA can be used toidentify its complement and, since DNA is double stranded, itsequivalent or homolog, using nucleic acid hybridization techniques. Italso will be recognized that hybridization can occur with less than 100%complementarity. However, given appropriate choice of conditions,hybridization techniques can be used to differentiate among DNAsequences based on their structural relatedness to a particular probe.For guidance regarding such conditions see, Sambrook et al., 1989 supraand Ausubel et al., 1995 (Ausubel, F. M., Brent, R., Kingston, R. E.,Moore, D. D., Sedman, J. G., Smith, J. A., & Struhl, K. eds. (1995).Current Protocols in Molecular Biology. New York: John Wiley and Sons).

Structural similarity between two polynucleotide sequences can beexpressed as a function of “stringency” of the conditions under whichthe two sequences will hybridize with one another. As used herein, theterm “stringency” refers to the extent that the conditions disfavorhybridization. Stringent conditions strongly disfavor hybridization, andonly the most structurally related molecules will hybridize to oneanother under such conditions. Conversely, non-stringent conditionsfavor hybridization of molecules displaying a lesser degree ofstructural relatedness. Hybridization stringency, therefore, directlycorrelates with the structural relationships of two nucleic acidsequences.

Hybridization stringency is a function of many factors, includingoverall DNA concentration, ionic strength, temperature, probe size andthe presence of agents which disrupt hydrogen bonding. Factors promotinghybridization include high DNA concentrations, high ionic strengths, lowtemperatures, longer probe size and the absence of agents that disrupthydrogen bonding. Hybridization typically is performed in two phases:the “binding” phase and the “washing” phase.

Functionally Equivalent DNA Variants

Yet another class of DNA variants within the scope of the invention maybe described with reference to the product they encode. Thesefunctionally equivalent polynucleotides are characterized by the factthat they encode the same peptide sequences due to the degeneracy of thegenetic code.

It is recognized that variants of DNA molecules provided herein can beconstructed in several different ways. For example, they may beconstructed as completely synthetic DNAs. Methods of efficientlysynthesizing oligonucleotides are widely available. See Ausubel et al.,section 2.11, Supplement 21 (1993). Overlapping oligonucleotides may besynthesized and assembled in a fashion first reported by Khorana et al.,J. Mol. Biol. 72:209-217 (1971); see also Ausubel et al., supra, Section8.2. Synthetic DNAs preferably are designed with convenient restrictionsites engineered at the 5′ and 3′ ends of the gene to facilitate cloninginto an appropriate vector.

As indicated, a method of generating variants is to start with one ofthe DNAs disclosed herein and then to conduct site-directed mutagenesis.See Ausubel et al., supra, chapter 8, Supplement 37 (1997). In a typicalmethod, a target DNA is cloned into a single-stranded DNA bacteriophagevehicle. Single-stranded DNA is isolated and hybridized with anoligonucleotide containing the desired nucleotide alteration(s). Thecomplementary strand is synthesized and the double stranded phage isintroduced into a host. Some of the resulting progeny will contain thedesired mutant, which can be confirmed using DNA sequencing. Inaddition, various methods are available that increase the probabilitythat the progeny phage will be the desired mutant. These methods arewell known to those in the field and kits are commercially available forgenerating such mutants.

Recombinant DNA Constructs and Expression

The present invention further provides recombinant DNA constructscomprising one or more of the nucleotide sequences encoding thepreferred antibodies of the present invention. The recombinantconstructs of the present invention can be used in connection with avector, such as a plasmid, phagemid, phage or viral vector, into which aDNA molecule encoding an antibody of the invention or antigen-bindingfragment thereof or variant thereof is inserted.

An antibody, antigen binding portion, or variant thereof provided hereincan be prepared by recombinant expression of nucleic acid sequencesencoding light and heavy chains or portions thereof in a host cell. Toexpress an antibody, antigen binding portion, or variant thereofrecombinantly a host cell can be transfected with one or morerecombinant expression vectors carrying DNA fragments encoding the lightand/or heavy chains or portions thereof such that the light and heavychains are expressed in the host cell. Standard recombinant DNAmethodologies are used to prepare and/or obtain nucleic acids encodingthe heavy and light chains, incorporate these nucleic acids intorecombinant expression vectors and introduce the vectors into hostcells, such as those described in Sambrook, Fritsch and Maniatis (eds.),Molecular Cloning; A Laboratory Manual, Second Edition, Cold SpringHarbor, N.Y., (1989), Ausubel, F. M. et al. (eds.) Current Protocols inMolecular Biology, Greene Publishing Associates, (1989) and in U.S. Pat.No. 4,816,397 by Boss et al.

In addition, the nucleic acid sequences encoding variable regions of theheavy and/or light chains can be converted, for example, to nucleic acidsequences encoding full-length antibody chains, Fab fragments, or toscFv. The VL- or VH-encoding DNA fragment can be operatively linked,(such that the amino acid sequences encoded by the two DNA fragments arein-frame) to another DNA fragment encoding, for example, an antibodyconstant region or a flexible linker. The sequences of human heavy chainand light chain constant regions are known in the art (see e.g., Kabat,E. A., el al. (1991) Sequences of Proteins of Immunological Interest,Fifth Edition, U.S. Department of Health and Human Services, NIHPublication No. 91-3242) and DNA fragments encompassing these regionscan be obtained by standard PCR amplification.

To create a polynucleotide sequence that encodes a scFv, the VH- andVL-encoding nucleic acids can be operatively linked to another fragmentencoding a flexible linker such that the VH and VL sequences can beexpressed as a contiguous single-chain protein, with the VL and VHregions joined by the flexible linker (see e.g., Bird et al. (1988)Science 242:423-426; Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883; McCafferty et al., Nature (1990) 348:552-554).

To express the antibodies, antigen binding fragments thereof or variantsthereof standard recombinant DNA expression methods can be used (see,for example, Goeddel; Gene Expression Technology. Methods in Enzymology185, Academic Press, San Diego, Calif. (1990)). For example, DNAencoding the desired polypeptide can be inserted into an expressionvector which is then transfected into a suitable host cell. Suitablehost cells are prokaryotic and eukaryotic cells. Examples forprokaryotic host cells are e.g. bacteria, examples for eukaryotic hostscells are yeasts, insects and insect cells, plants and plant cells,transgenic animals, or mammalian cells. In some embodiments, the DNAsencoding the heavy and light chains are inserted into separate vectors.In other embodiments, the DNA encoding the heavy and light chains isinserted into the same vector. It is understood that the design of theexpression vector, including the selection of regulatory sequences isaffected by factors such as the choice of the host cell, the level ofexpression of protein desired and whether expression is constitutive orinducible.

Therefore, an embodiment of the present invention are also host cellscomprising the vector or a nucleic acid molecule, whereby the host cellcan be a higher eukaryotic host cell, such as a mammalian cell, a lowereukaryotic host cell, such as a yeast cell, and may be a prokaryoticcell, such as a bacterial cell.

Another embodiment of the present invention is a method of using thehost cell to produce an antibody and antigen binding fragments,comprising culturing the host cell under suitable conditions andrecovering said antibody.

Therefore another embodiment of the present invention is the productionof the antibodies according to this invention with the host cells of thepresent invention and purification of these antibodies to at least 95%homogeneity by weight.

Bacterial Expression

Useful expression vectors for bacterial use are constructed by insertinga DNA sequence encoding a desired protein together with suitabletranslation initiation and termination signals in operable reading phasewith a functional promoter. The vector will comprise one or morephenotypic selectable markers and an origin of replication to ensuremaintenance of the vector and, if desirable, to provide amplificationwithin the host. Suitable prokaryotic hosts for transformation includebut are not limited to E. coli, Bacillus subtilis, Salmonellatyphimurium and various species within the genera Pseudomonas,Streptomyces, and Staphylococcus.

Bacterial vectors may be, for example, bacteriophage-, plasmid- orphagemid-based. These vectors can contain a selectable marker and abacterial origin of replication derived from commercially availableplasmids typically containing elements of the well-known cloning vectorpBR322 (ATCC 37017). Following transformation of a suitable host strainand growth of the host strain to an appropriate cell density, theselected promoter is de-repressed/induced by appropriate means (e.g.,temperature shift or chemical induction) and cells are cultured for anadditional period. Cells are typically harvested by centrifugation,disrupted by physical or chemical means, and the resulting crude extractretained for further purification.

In bacterial systems, a number of expression vectors may beadvantageously selected depending upon the use intended for the proteinbeing expressed. For example, when a large quantity of such a protein isto be produced, for the generation of antibodies or to screen peptidelibraries, for example, vectors which direct the expression of highlevels of fusion protein products that are readily purified may bedesirable.

Therefore, an embodiment of the present invention is an expressionvector comprising a nucleic acid sequence encoding for the novelantibodies of the present invention.

Antibodies of the present invention or antigen-binding fragments thereofor variants thereof include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from a prokaryotic host, including, for example, E. coli,Bacillus subtilis, Salmonella typhimurium and various species within thegenera Pseudomonas, Streptomyces, and Staphylococcus, preferably, fromE. coli cells.

Mammalian Expression

Preferred regulatory sequences for mammalian host cell expressioninclude viral elements that direct high levels of protein expression inmammalian cells, such as promoters and/or enhancers derived fromcytomegalovirus (CMV) (such as the CMV promoter/enhancer), Simian Virus40 (SV40) (such as the SV40 promoter/enhancer), adenovirus, (e.g., theadenovirus major late promoter (AdMLP)) and polyoma. Expression of theantibodies may be constitutive or regulated (e.g. inducible by additionor removal of small molecule inductors such as Tetracyclin inconjunction with Tet system). For further description of viralregulatory elements, and sequences thereof, see e.g., U.S. Pat. No.5,168,062 by Stinski, U.S. 4,510,245 by Bell et al. and U.S. Pat. No.4,968,615 by Schaffner et al. The recombinant expression vectors canalso include origins of replication and selectable markers (see e.g.,U.S. Pat. Nos. 4,399,216, 4,634,665 and U.S. Suitable selectable markersinclude genes that confer resistance to drugs such as G418, puromycin,hygromycin, blasticidin, zeocin/bleomycin or methotrexate or selectablemarker that exploit auxotrophies such as Glutamine Synthetase(Bebbington et al., Biotechnology (N Y). 1992 February; 10(2):169-75),on a host cell into which the vector has been introduced. For example,the dihydrofolate reductase (DHFR) gene confers resistance tomethotrexate, neo gene confers resistance to G418, the bsd gene fromAspergillus terreus confers resistance to blasticidin, puromycinN-acetyl-transferase confers resistance to puromycin, the Sh ble geneproduct confers resitance to zeocin, and resistance to hygromycin isconferred by the E. coli hygromycin resistance gene (hyg or hph).Selectable markers like DHFR or Glutamine Synthetase are also useful foramplification techniques in conjunction with MTX and MSX.

Transfection of the expression vector into a host cell can be carriedout using standard techniques such as electroporation, nucleofection,calcium-phosphate precipitation, lipofection, polycation-basedtransfection such as polyethlylenimine (PEI)-based transfection andDEAE-dextran transfection.

Suitable mammalian host cells for expressing the antibodies, antigenbinding fragments thereof or variants thereof provided herein includeChinese Hamster Ovary (CHO cells) such as CHO-K1, CHO-S, CHO-K1SV[including dhfr-CHO cells, described in Urlaub and ChasM, (1980) Proc.Natl. Acad. Sci. USA 77:4216-4220 and Urlaub et al., Cell. 1983June;33(2):405-12, used with a DHFR selectable marker, e.g., asdescribed in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621; and other knockout cells exemplified in Fan et al.,Biotechnol Bioeng. 2012 April; 109(4):1007-15], NSO myeloma cells, COScells, HEK293 cells, HKB11 cells, BHK21 cells, CAP cells, EB66 cells,and SP2 cells.

Expression might also be transient or semi-stable in expression systemssuch as HEK293, HEK293T, HEK293-EBNA, HEK293E, HEK293-6E,HEK293-Freestyle, HKB11, Expi293F, 293EBNALT75, CHO Freestyle, CHO-S,CHO-K1, CHO-K1SV, CHOEBNALT85, CHOS-XE, CHO—3E7 or CAP-T cells (forinstance Durocher et al., Nucleic Acids Res. 2002 Jan. 15; 30(2):E9).

In some embodiments, the expression vector is designed such that theexpressed protein is secreted into the culture medium in which the hostcells are grown. The antibodies, antigen binding fragments thereof orvariants thereof can be recovered from the culture medium using standardprotein purification methods.

Purification

Antibodies of the invention or antigen-binding fragments thereof orvariants thereof can be recovered and purified from recombinant cellcultures by well-known methods including, but not limited to ammoniumsulfate or ethanol precipitation, acid extraction, Protein Achromatography, Protein G chromatography, anion or cation exchangechromatography, phospho-cellulose chromatography, hydrophobicinteraction chromatography, affinity chromatography, hydroxylapatitechromatography and lectin chromatography. High performance liquidchromatography (“HPLC”) can also be employed for purification. See,e.g., Colligan, Current Protocols in Immunology, or Current Protocols inProtein Science, John Wiley & Sons, NY, N.Y., (1997-2001), e.g.,Chapters 1, 4, 6, 8, 9, 10, each entirely incorporated herein byreference.

Antibodies of the present invention or antigen-binding fragments thereofor variants thereof include naturally purified products, products ofchemical synthetic procedures, and products produced by recombinanttechniques from an eukaryotic host, including, for example, yeast,higher plant, insect and mammalian cells. Depending upon the hostemployed in a recombinant production procedure, the antibody of thepresent invention can be glycosylated or can be non-glycosylated. Suchmethods are described in many standard laboratory manuals, such asSambrook, supra, Sections 17.37-17.42; Ausubel, supra, Chapters 10, 12,13, 16, 18 and 20.

In preferred embodiments, the antibody is purified (1) to greater than95% by weight of antibody as determined e.g. by the Lowry method, UV-Visspectroscopy or by by SDS-Capillary Gel electrophoresis (for example ona Caliper LabChip GXII, GX 90 or Biorad Bioanalyzer device), and infurther preferred embodiments more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence, or (3) to homogeneity by SDS-PAGE under reducing ornon-reducing conditions using Coomassie blue or, preferably, silverstain. Isolated naturally occurring antibody includes the antibody insitu within recombinant cells since at least one component of theantibody's natural environment will not be present. Ordinarily, however,isolated antibody will be prepared by at least one purification step.

Isotopes, Salts, Solvates, Isotopic Variants

The present invention also encompasses all suitable isotopic variants ofthe compounds according to the invention. An isotopic variant of acompound according to the invention is understood here as meaning acompound in which at least one atom within the compound according to theinvention has been exchanged for another atom of the same atomic number,but with a different atomic mass than the atomic mass which usually orpredominantly occurs in nature. Examples of isotopes which can beincorporated into a compound according to the invention are those ofhydrogen, carbon, nitrogen, oxygen, phosphorus, sulphur, fluorine,chlorine, bromine and iodine, such as ²H (deuterium), ³H (tritium), ¹³C,¹⁴C, ¹⁵N, ¹⁷O, ¹⁸O, ³²P, ³³P, ³³S, ³⁴S, ³⁵S, ³⁶S, ¹⁸F, ³⁶Cl, ⁸²Br, ¹²³I,¹²⁴I, ¹²⁹I, and ¹³¹I. Particular isotopic variants of a compoundaccording to the invention, especially those in which one or moreradioactive isotopes have been incorporated, may be beneficial, forexample, for the examination of the mechanism of action or of the activecompound distribution in the body; due to comparatively easypreparability and detectability, especially compounds labelled with ³Hor ¹⁴C isotopes are suitable for this purpose. In addition, theincorporation of isotopes, for example of deuterium, can lead toparticular therapeutic benefits as a consequence of greater metabolicstability of the compound, for example an extension of the half-life inthe body or a reduction in the active dose required; such modificationsof the compounds according to the invention may therefore in some casesalso constitute a preferred embodiment of the present invention.Isotopic variants of the compounds according to the invention can beprepared by the processes known to those skilled in the art, for exampleby the methods described below and the procedures described in theworking examples, by using corresponding isotopic modifications of therespective reagents and/or starting compounds.

Preferred salts in the context of the present invention arephysiologically acceptable salts of the compounds according to theinvention. Also encompassed are salts which are not themselves suitablefor pharmaceutical applications but can be used, for example, forisolation or purification of the compounds according to the invention.

Physiologically acceptable salts of the compounds according to theinvention include acid addition salts of mineral acids, carboxylic acidsand sulphonic acids, for example salts of hydrochloric acid, hydrobromicacid, sulphuric acid, phosphoric acid, methanesulphonic acid,ethanesulphonic acid, benzenesulphonic acid, toluenesulphonic acid,naphthalenedisulphonic acid, acetic acid, trifluoroacetic acid,propionic acid, lactic acid, tartaric acid, malic acid, citric acid,fumaric acid, maleic acid and benzoic acid.

Physiologically acceptable salts of the inventive compounds also includesalts of conventional bases, by way of example and with preferencealkali metal salts (e.g. sodium and potassium salts), alkaline earthmetal salts (e.g. calcium and magnesium salts) and ammonium saltsderived from ammonia or organic amines having 1 to 16 carbon atoms, byway of example and with preference ethylamine, diethylamine,triethylamine, ethyldiisopropylamine, monoethanolamine, diethanolamine,triethanolamine, dicyclohexylamine, dimethylaminoethanol, procaine,dibenzylamine, N-methylpiperidine, N-methylmorpholine, arginine, lysineand 1,2-ethylenediamine.

Solvates in the context of the invention are described as those forms ofthe compounds according to the invention which form a complex in thesolid or liquid state by coordination with solvent molecules. Hydratesare a specific form of the solvates in which the coordination is withwater. Solvates preferred in the context of the present invention arehydrates.

In addition, the present invention also encompasses prodrugs of thecompounds according to the invention. The term “prodrugs” here denotescompounds which may themselves be biologically active or inactive, butare converted (for example by metabolic or hydrolytic means) toinventive compounds during their residence time in the body.

Another embodiment of the invention is a conjugate as defined abovewhere the antibody or functional fragment thereof binds to a cancertarget molecule.

Another embodiment of the invention is a conjugate as defined abovewhere the conjugate has 2 to 6 conjugation sites per antibody orfunctional fragment thereof.

Another embodiment of the invention is a conjugate as defined abovewhere the conjugate has 2 conjugation sites per antibody or functionalfragment thereof.

Another embodiment of the invention is a conjugate according as definedabove where the conjugate has 4 conjugation sites per antibody orfunctional fragment thereof.

Another embodiment of the invention is a conjugate as defined abovewhere the antibody or functional fragment thereof binds to anextracellular target molecule.

Another embodiment of the invention is a conjugate as defined abovewhere the antibody or functional fragment thereof, after binding to theextracellular target molecule, is internalized and processedintracellularly (preferably lysosomally) by the cell expressing thetarget molecule.

Therapeutic Use

The CD123 targeted antibody-drug conjugates described herein may be usedto treat a CD123 expressing disorder, such as CD 123 expressing cancer.Typically such cancers show detectable levels of CD123 measured at theprotein (e.g., by immunoassay) or RNA level. Some such cancers showelevated levels of CD123 relative to noncancerous tissue of the sametype, preferably from the same patient. Optionally, a level of CD 123 ina cancer is measured before performing treatment.

The CD 123 directed antibody drug conjugates may be used to treat CD123expressing disease, including CD123 expressing cancers, like tumors ofthe hematopoietic and lymphoid tissues or hematopoietic and lymphoidmalignancies.

Examples of cancers associated with CD123 expression include myeloiddiseases such as, acute myeloid leukemia (AML) and myelodysplasticsyndrome (MDS). Other cancers include B-cell acute lymphoblasticleukemia (B-ALL), hairy cell leukemia, Fanconi anemia, Blasticplasmacytoid dendritic cell neoplasm (BPDCN), Hodgkin's disease,Immature T-cell acute lymphoblastic leukemia (Immature T-ALL), Burkitt'slymphoma, Follicular lymphoma, chronic lymphocytic leukemia (CLL), ormantle cell lymphoma.

Methods of the present invention include treating a patient that has acancer that expresses CD123 comprising administering to the patient anantibody-drug conjugate of the present invention. The cancer can be anyCD123 expressing cancer, including, for example, AML, MDS, B-ALL, hairycell leukemia, Fanconi anemia, BPDCN, Hodgkin's disease, Immature T-ALL,Burkitt's lymphoma, Follicular lymphoma, CLL, or mantle cell lymphoma.

The hyper-proliferative diseases, for the treatment of which thecompounds according to the invention may be employed, include inparticular the group of cancer and tumour diseases. In the context ofthe present invention, these are understood to mean especially thefollowing diseases, but without any limitation thereto: mammarycarcinomas and mammary tumours (mammary carcinomas including ductal andlobular forms, also in situ), tumours of the respiratory tract(small-cell and non-small cell carcinoma, bronchial carcinoma), cerebraltumours (e.g. of the brain stem and of the hypothalamus, astrocytoma,ependymoma, glioblastoma, glioma, medulloblastoma, meningioma andneuro-ectormal and pineal tumours), tumours of the digestive organs(carcinomas of the oesophagus, stomach, gall bladder, small intestine,large intestine, rectum and anal carcinomas), liver tumours (inter aliahepatocellular carcinoma, cholangiocarcinoma and mixed hepatocellularcholangiocarcinoma), tumours of the head and neck region (larynx,hypopharynx, nasopharynx, oropharynx, lips and oral cavity carcinomas,oral melanomas), skin tumours (basaliomas, spinaliomas, squamous cellcarcinomas, Kaposi's sarcoma, malignant melanoma, non-melanomatous skincancer, Merkel cell skin cancer, mast cell tumours), tumours of softtissue (inter alia soft tissue sarcomas, osteosarcomas, malignantfibrous histiocytomas, chondrosarcomas, fibrosarcomas, hemangiosarcomas,leiomyosarcomas, liposarcomas, lymphosarcomas and rhabdomyosarcomas),tumours of the eyes (inter alia intraocular melanoma andretinoblastoma), tumours of the endocrine and exocrine glands (e.g. ofthe thyroid and parathyroid glands, pancreas and salivary glandcarcinomas, adenocarcinomas), tumours of the urinary tract (tumours ofthe bladder, penis, kidney, renal pelvis and ureter) and tumours of thereproductive organs (carcinomas of the endometrium, cervix, ovary,vagina, vulva and uterus in women and carcinomas of the prostate andtestes in men). These also include proliferative blood diseases of theblood, the lymph system and the spinal cord, in solid form and ascirculating cells, such as leukaemias, lymphomas and myeloproliferativediseases, for example acute myeloid, acute lymphoblastic, chroniclymphocytic, chronic myelogenous and hairy cell leukaemia, andAIDS-correlated lymphomas, Hodgkin's lymphomas, non-Hodgkin's lymphomas,cutaneous T cell lymphomas, Burkitt's lymphomas and lymphomas in thecentral nervous system.

These well-characterized diseases in humans can also occur with acomparable aetiology in other mammals and can likewise be treated therewith the compounds of the present invention.

The treatment of the cancer diseases mentioned above with the compoundsaccording to the invention comprises both a treatment of the solidtumors and a treatment of metastasizing or circulating forms thereof.

In the context of this invention, the term “treatment” or “treat” isused in the conventional sense and means attending to, caring for andnursing a patient with the aim of combating, reducing, attenuating oralleviating a disease or health abnormality, and improving the livingconditions impaired by this disease, as, for example, in the event of acancer.

The present invention thus further provides for the use of the compoundsaccording to the invention for the treatment and/or prevention ofdisorders, in particular the disorders mentioned above.

The present invention further provides for the use of the compoundsaccording to the invention for producing a medicament for the treatmentand/or prevention of disorders, in particular the disorders mentionedabove.

The present invention further provides for the use of the compoundsaccording to the invention in a method for treatment and/or preventionof disorders, in particular the disorders mentioned above.

The present invention further provides a method for treatment and/orprevention of disorders, in particular the disorders mentioned above,using an effective amount of at least one of the compounds according tothe invention.

The compounds according to the invention can be used alone or, ifrequired, in combination with one or more other pharmacologically activesubstances, provided that this combination does not lead to undesirableand unacceptable side effects. The present invention furthermoretherefore provides medicaments containing at least one of the compoundsaccording to the invention and one or more further active compounds, inparticular for treatment and/or prevention of the abovementioneddisorders.

For example, the compounds of the present invention can be combined withknown anti-hyper-proliferative, cytostatic or cytotoxic substances forthe treatment of cancer diseases. Examples of suitable combinationactive compounds include:

131I-chTNT, abarelix, abiraterone, aclarubicin, ado-trastuzumabemtansine, afatinib, aflibercept, aldesleukin, alemtuzumab, Alendronicacid, alitretinoin, altretamine, amifostine, aminoglutethimide, Hexylaminolevulinate, amrubicin, amsacrine, anastrozole, ancestim, anetholedithiolethione, angiotensin II, antithrombin III, aprepitant,arcitumomab, arglabin, arsenic trioxide, asparaginase, axitinib,azacitidine, basiliximab, belotecan, bendamustine, belinostat,bevacizumab, bexarotene, bicalutamide, bisantrene, bleomycin,bortezomib, buserelin, bosutinib, brentuximab vedotin, busulfan,cabazitaxel, cabozantinib, calcium folinate, calcium levofolinate,capecitabine, capromab, carboplatin, carfilzomib, carmofur, carmustine,catumaxomab, celecoxib, celmoleukin, ceritinib, cetuximab, chlorambucil,chlormadinone, chlormethine, cidofovir, cinacalcet, cisplatin,cladribine, clodronic acid, clofarabine, copanlisib, crisantaspase,cyclophosphamide, cyproterone, cytarabine, dacarbazine, dactinomycin,darbepoetin alfa, dabrafenib, dasatinib, daunorubicin, decitabine,degarelix, denileukin diftitox, denosumab, depreotide, deslorelin,dexrazoxane, dibrospidium chloride, dianhydrogalactitol, diclofenac,docetaxel, dolasetron, doxifluridine, doxorubicin, doxorubicin+estrone,dronabinol, eculizumab, edrecolomab, elliptinium acetate, eltrombopag,endostatin, enocitabine, enzalutamide, epirubicin, epitiostanol, epoetinalfa, epoetin beta, epoetin zeta, eptaplatin, eribulin, erlotinib,esomeprazole, estradiol, estramustine, etoposide, everolimus,exemestane, fadrozole, fentanyl, filgrastim, fluoxymesterone,floxuridine, fludarabine, fluorouracil, flutamide, folinic acid,formestane, fosaprepitant, fotemustine, fulvestrant, gadobutrol,gadoteridol, gadoteric acid meglumine, gadoversetamide, gadoxetic acid,gallium nitrate, ganirelix, gefitinib, gemcitabine, gemtuzumab,Glucarpidase, glutoxim, GM-CSF, goserelin, granisetron, granulocytecolony stimulating factor, histamine dihydrochloride, histrelin,hydroxycarbamide, 1-125 seeds, lansoprazole, ibandronic acid,ibritumomab tiuxetan, ibrutinib, idarubicin, ifosfamide, imatinib,imiquimod, improsulfan, indisetron, incadronic acid, ingenol mebutate,interferon alfa, interferon beta, interferon gamma, iobitridol,iobenguane (123I), iomeprol, ipilimumab, irinotecan, Itraconazole,ixabepilone, lanreotide, lapatinib, Iasocholine, lenalidomide,lenograstim, lentinan, letrozole, leuprorelin, levamisole,levonorgestrel, levothyroxine sodium, lisuride, lobaplatin, lomustine,lonidamine, masoprocol, medroxyprogesterone, megestrol, melarsoprol,melphalan, mepitiostane, mercaptopurine, mesna, methadone, methotrexate,methoxsalen, methylaminolevulinate, methylprednisolone,methyltestosterone, metirosine, mifamurtide, miltefosine, miriplatin,mitobronitol, mitoguazone, mitolactol, mitomycin, mitotane,mitoxantrone, mogamulizumab, molgramostim, mopidamol, morphinehydrochloride, morphine sulfate, nabilone, nabiximols, nafarelin,naloxone+pentazocine, naltrexone, nartograstim, nedaplatin, nelarabine,neridronic acid, nivolumabpentetreotide, nilotinib, nilutamide,nimorazole, nimotuzumab, nimustine, nitracrine, nivolumab, obinutuzumab,octreotide, ofatumumab, omacetaxine mepesuccinate, omeprazole,ondansetron, oprelvekin, orgotein, orilotimod, oxaliplatin, oxycodone,oxymetholone, ozogamicine, p53 gene therapy, paclitaxel, palifermin,palladium-103 seed, palonosetron, pamidronic acid, panitumumab,pantoprazole, pazopanib, pegaspargase, PEG-epoetin beta (methoxyPEG-epoetin beta), pembrolizumab, pegfilgrastim, peginterferon alfa-2b,pemetrexed, pentazocine, pentostatin, peplomycin, Perflubutane,perfosfamide, Pertuzumab, picibanil, pilocarpine, pirarubicin,pixantrone, plerixafor, plicamycin, poliglusam, polyestradiol phosphate,polyvinylpyrrolidone+sodium hyaluronate, polysaccharide-K, pomalidomide,ponatinib, porfimer sodium, pralatrexate, prednimustine, prednisone,procarbazine, procodazole, propranolol, quinagolide, rabeprazole,racotumomab, radium-223 chloride, radotinib, raloxifene, raltitrexed,ramosetron, ramucirumab, ranimustine, rasburicase, razoxane,refametinib, regorafenib, risedronic acid, rhenium-186 etidronate,rituximab, romidepsin, romiplostim, romurtide, roniciclib, samarium(153Sm) lexidronam, sargramostim, satumomab, secretin, sipuleucel-T,sizofiran, sobuzoxane, sodium glycididazole, sorafenib, stanozolol,streptozocin, sunitinib, talaporfin, tamibarotene, tamoxifen,tapentadol, tasonermin, teceleukin, technetium (99mTc) nofetumomabmerpentan, 99mTc-HYNIC-ITyr31-octreotide, tegafur,tegafur+gimeracil+oteracil, temoporfin, temozolomide, temsirolimus,teniposide, testosterone, tetrofosmin, thalidomide, thiotepa,thymalfasin, thyrotropin alfa, tioguanine, tocilizumab, topotecan,toremifene, tositumomab, trabectedin, tramadol, trastuzumab, trastuzumabemtansine, treosulfan, tretinoin, trifluridine+tipiracil, trilostane,triptorelin, trametinib, trofosfamide, thrombopoietin, tryptophan,ubenimex, valatinib, valrubicin, vandetanib, vapreotide, vemurafenib,vinblastine, vincristine, vindesine, vinflunine, vinorelbine,vismodegib, vorinostat, vorozole, yttrium-90 glass microspheres,zinostatin, zinostatin stimalamer, zoledronic acid, zorubicin.

In addition, the compounds of the present invention can be combined, forexample, with binders which, by way of example, can bind to thefollowing targets: OX-40, CD137/4-1BB, DR3, IDO1/IDO2, LAG-3, CD40.

In addition, the compounds according to the invention can also be usedin combination with radiotherapy and/or surgical intervention.

Generally, the following aims can be pursued with the combination ofcompounds of the present invention with other cytostatically orcytotoxically active agents:

-   -   improved efficacy in slowing the growth of a tumour, in reducing        its size or even in the complete elimination thereof, compared        with treatment with an individual active compound;    -   the possibility of using the chemotherapeutics used in a lower        dosage than in the case of monotherapy;    -   the possibility of a more tolerable therapy with fewer side        effects compared with individual administration;    -   the possibility of treatment of a broader spectrum of tumour        diseases;    -   the achievement of a higher rate of response to the therapy;    -   a longer survival time of the patient compared with present-day        standard therapy.

In addition, the compounds according to the invention can also be usedin combination with radiotherapy and/or surgical intervention.

The present invention further provides medicaments which comprise atleast one compound according to the invention, typically together withone or more inert, nontoxic, pharmaceutically suitable excipients, andthe use thereof for the aforementioned purposes.

The compounds according to the invention can act systemically and/orlocally. For this purpose, they can be administered in a suitablemanner, for example parenterally, possibly inhalatively or as implantsor stents.

The compounds according to the invention can be administered in suitableadministration forms for these administration routes.

Parenteral administration can bypass an absorption step (for exampleintravenously, intraarterially, intracardially, intraspinally orintralumbally) or include an absorption (for example intramuscularly,subcutaneously, intracutaneously, percutaneously or intraperitoneally).Administration forms suitable for parenteral administration includepreparations for injection and infusion in the form of solutions,suspensions, emulsions or lyophilizates. Preference is given toparenteral administration, especially intravenous administration.

In general, it has been found to be advantageous in the case ofparenteral administration to administer amounts of from about 0.001 to 1mg/kg, preferably about 0.01 to 0.5 mg/kg, of body weight to achieveeffective results.

It may nevertheless be necessary where appropriate to deviate from thestated amounts, specifically as a function of body weight, route ofadministration, individual response to the active compound, nature ofthe preparation and time or interval over which administration takesplace. Thus, in some cases less than the abovementioned minimum amountmay be sufficient, while in other cases the upper limit mentioned mustbe exceeded. In the case of administration of greater amounts, it may beadvisable to divide them into several individual doses over the day.

EXAMPLES

The examples which follow illustrate the invention. The invention is notrestricted to the examples.

Unless stated otherwise, the percentages in the tests and examples whichfollow are percentages by weight; parts are parts by weight. Solventratios, dilution ratios and concentration data for the liquid/liquidsolutions are based in each case on volume.

If, in the description of experiments, the temperature at which thereaction is carried out is not stated, room temperature can be assumed.

Synthesis routes:

Exemplary for the working examples, the schemes below show exemplarysynthesis routes leading to the working examples:

Exemplary for the working examples, the schemes below show exemplarysynthesis routes leading to the working examples:

A. Examples Abbreviations and Acronyms

-   -   ABCB1 ATP-binding cassette sub-family B member 1 (synonym for        P-gp and MDR1)    -   ATP adenosine triphosphate    -   BCRP breast cancer resistance protein, an efflux transporter    -   BEP 2-bromo-1-ethylpyridinium tetrafluoroborate    -   d doublet (in NMR)    -   TLC thin-layer chromatography    -   DCM dichloromethane    -   dd doublet of doublets (in NMR)    -   DMF N,N-dimethylformamide    -   DMSO dimethyl sulphoxide    -   DPBS, D-PBS, PBS Dulbecco's phosphate-buffered salt solution        -   PBS=DPBS=D-PBS, pH 7.4, from Sigma, No D8537        -   Composition:        -   0.2 g KCl        -   0.2 g KH₂PO₄ (anhyd)        -   8.0 g NaCl        -   1.15 g Na₂HPO₄ (anhyd)        -   made up ad 1 l with H₂O    -   DTT DL-dithiothreitol    -   EDC N′-(3-dimethylaminopropyl)-N-ethylearbodiimide hydrochloride    -   EI electron impact ionization (in MS)    -   ELISA enzyme-linked immunosorbent assay    -   ESI electrospray ionization (in MS)    -   ESI-MicroTofq ESI-MicroTofq (name of the mass spectrometer with        Tof=time of flight and q=quadrupol)    -   FCS foetal calf serum    -   Fmoc (9H-fluoren-9-ylmethoxy)carbonyl    -   sat. saturated    -   GTP guanosine-5′-triphosphate    -   h hour(s)    -   HATU 0-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium        hexafluorophosphate    -   HEPES 4-(2-hydroxyethyl)piperazine-1-ethanesulphonic acid    -   HPLC high-pressure, high-performance liquid chromatography    -   IC₅₀ half-maximal inhibitory concentration    -   i.v. intravenously, administration into the vein    -   KG-1 human tumour cell line    -   LC-MS liquid chromatography-coupled mass spectrometry    -   LLC-PK1 cells Lewis lung carcinoma pork kidney cell line    -   L-MDR human MDR1 transfected LLC-PK1 cells    -   LoVo human tumour cell line    -   m multiplet (in NMR)    -   MDR1 Multidrug resistance protein 1    -   MeCN acetonitrile    -   min minute(s)    -   MOLM-13 human tumour cell line    -   MS mass spectrometry    -   MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium        bromide 3    -   MV-4-11 human tumour cell line    -   NCI-H292 human tumour cell line    -   NMR nuclear magnetic resonance spectrometry    -   NMRI mouse strain originating from the Naval Medical Research        Institute (NMRI)    -   NB4 human tumour cell line    -   PBS phosphate-buffered salt solution    -   P-gp P-gycoprotein, a transporter protein    -   PNGaseF enzyme for cleaving sugar    -   RT room temperature    -   R t retention time (in HPLC)    -   s singlet (in NMR)    -   SCID mice test mice with severe combined immunodeficiency    -   t triplet (in NMR)    -   TEMPO (2,2,6,6-tetramethylpiperidin-1-yl)oxyl    -   tert Tertiary    -   TFA trifluoroacetic acid    -   THF Tetrahydrofuran    -   THP-1 human tumour cell line    -   UV ultraviolet spectrometry    -   Z Benzyloxycarbonyl

HPLC and LC-MS methods:

Method 1 (LC-MS):

Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLCHSS T3 1.8μ 50×1 mm; mobile phase A: 1 l of water+0.25 ml of 99%strength formic acid, mobile phase B: 1 l of acetonitrile+0.25 ml of 99%strength formic acid; gradient: 0.0 min 90% A→1.2 min 5% A→2.0 min 5% Aoven: 50° C.; flow rate: 0.40 ml/min; UV detection: 208-400 nm.

Method 2 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: WatersAcquity I-CLASS; column: Waters, BEH300, 2.1×150 mm, C18 1.7 μm; mobilephase A: 1 l of water+0.01% formic acid; mobile phase B: 1 l ofacetonitrile+0.01% formic acid; gradient: 0.0 min 2% B→1.5 min 2% B→8.5min 95% B→10.0 min 95% B; oven: 50° C.; flow rate: 0.50 ml/min; UVdetection: 220 nm

Method 3 (LC-MS):

MS instrument: Waters (Micromass) QM; HPLC instrument: Agilent 1100Series; column: Agilent ZORBAX Extend-C18 3.0×50 mm 3.5-micron; mobilephase A: 1 l of water+0.01 mol of ammonium carbonate, mobile phase B: 1l of acetonitrile; gradient: 0.0 min 98% A→0.2 min 98% A→3.0 min 5%A→4.5 min 5% A; oven: 40° C.; flow rate: 1.75 ml/min; UV detection: 210nm

Method 4 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: WatersAcquity I-CLASS; column: Waters, HSST3, 2.1×50 mm, C18 1.8 μm; mobilephase A: 1 l of water+0.01% formic acid; mobile phase B: 1 l ofacetonitrile+0.01% formic acid; gradient: 0.0 min 10% B→0.3 min 10%B→1.7 min 95% B→2.5 min 95% B; oven: 50° C.; flow rate: 1.20 ml/min; UVdetection: 210 nm

Method 5 (LC-MS):

Instrument: Waters ACQUITY SQD UPLC System; column: Waters Acquity UPLCHSS T3 1.8μ 50×1 mm; mobile phase A: 1 l of water+0.25 ml of 99%strength formic acid, mobile phase B: 1 l of acetonitrile+0.25 ml of 99%strength formic acid; gradient: 0.0 min 95% A→6.0 min 5% A→7.5 min 5% Aoven: 50° C.; flow rate: 0.35 ml/min; UV detection: 210-400 nm.

Method 6 (LC-MS):

Instrument: Micromass Quattro Premier with Waters UPLC Acquity; column:Thermo Hypersil GOLD 1.9μ 50×1 mm; mobile phase A: 1 l of water+0.5 mlof 50% strength formic acid, mobile phase B: 1 l of acetonitrile+0.5 mlof 50% strength formic acid; gradient: 0.0 min 97% A→0.5 min 97% A→3.2min 5% A→4.0 min 5% A oven: 50° C.; flow rate: 0.3 ml/min; UV detection:210 nm.

Method 7 (LC-MS):

Instrument: Agilent MS Quad 6150; HPLC: Agilent 1290; column: WatersAcquity UPLC HSS T3 1.8μ 50×2.1 mm; mobile phase A: 1 l of water+0.25 mlof 99% strength formic acid, mobile phase B: 1 l of acetonitrile+0.25 mlof 99% strength formic acid; gradient: 0.0 min 90% A→0.3 min 90% A→1.7min 5% A→3.0 min 5% A oven: 50° C.; flow rate: 1.20 ml/min; UVdetection: 205-305 nm.

Method 8 (LC-MS):

MS instrument type: Waters Synapt G2S; UPLC instrument type: WatersAcquity I-CLASS; column: Waters, HSST3, 2.1×50 mm, C18 1.8 μm; mobilephase A: 1 l of water+0.01% formic acid; mobile phase B: 1 l ofacetonitrile+0.01% formic acid; gradient: 0.0 min 2% B→2.0 min 2% B→13.0min 90% B→15.0 min 90% B; oven: 50° C.; flow rate: 1.20 ml/min; UVdetection: 210 nm

Method 9: LC-MS-Prep purification method for Examples 181-191 (MethodLIND-LC-MS-Prep)

MS instrument: Waters, HPLC instrument: Waters (column Waters X-BridgeC18, 19 mm×50 mm, 5 μm, mobile phase A: water+0.05% ammonia, mobilephase B: acetonitrile (ULC) with gradient; flow rate: 40 ml/min; UVdetection: DAD; 210⁻⁴⁰⁰ nm).

or

MS instrument: Waters, HPLC instrument: Waters (column Phenomenex Luna5μ C18(2) 100A, AXIA Tech. 50×21.2 mm, mobile phase A: water+0.05%formic acid, mobile phase B: acetonitrile (ULC) with gradient; flowrate: 40 ml/min; UV detection: DAD; 210⁻⁴⁰⁰ nm).

Method 10: LC-MS analysis method for Examples 181-191 (LIND_SQD_SB_AQ)

MS instrument: Waters SQD; Instrument HPLC: Waters UPLC; column: ZorbaxSB-Aq (Agilent), 50 mm×2.1 mm, 1.8 μm; mobile phase A: water+0.025%formic acid, mobile phase B: acetonitrile (ULC)+0.025% formic acid;gradient: 0.0 min 98% A−0.9 min 25% A−1.0 min 5% A−1.4 min 5% A−1.41 min98% A−1.5 min 98% A; oven: 40° C.; flow rate: 0.600 ml/min; UVdetection: DAD; 210 nm.

Method 11 (HPLC):

Instrument: HP1100 Series

column: Merck Chromolith SpeedROD RP-18e, 50-4.6 mm, Cat.

-   -   No. 1.51450.0001, precolumn Chromolith Guard Cartridge Kit,        RP-18e, 5-4.6 mm, Cat. No. 1.51470.0001

gradient: flow rate 5 ml/min

-   -   injection volume 5 μl

solvent A: HClO4 (70% strength) in water (4 ml/1)

solvent B: acetonitrile

start 20% B

0.50 min 20% B

3.00 min 90% B

3.50 min 90% B

3.51 min 20% B

4.00 min 20% B

column temperature: 40° C.

wavelength: 210 nm

Method 12 (LC-MS):

MS instrument type: Thermo Scientific FT-MS; UHPLC+instrument type:Thermo Scientific UltiMate 3000; column: Waters, HSST3, 2.1×75 mm, C181.8 μm; mobile phase A: 1 l of water+0.01% formic acid; mobile phase B:1 l of acetonitrile+0.01% formic acid; gradient: 0.0 min 10% B→2.5 min95% 30 B→3.5 min 95% B; oven: 50° C.; flow rate: 0.90 ml/min; UVdetection: 210 nm/optimum integration path 210⁻³⁰⁰ nm

Method 13: (LC-MS):

MS instrument: Waters (Micromass) Quattro Micro; Instrument Waters UPLCAcquity; column: Waters BEH C18 1.7 it 50×2.1 mm; mobile phase A: 1 l ofwater+0.01 mol ammonium formate, mobile phase

B: 1 l of acetonitrile; gradient: 0.0 min 95% A→0.1 min 95% A→2.0 min15% A→2.5 min 15% A→2.51 min 10% A→3.0 min 10% A; oven: 40° C.; flowrate: 0.5 ml/min; UV detection: 210 nm

All reactants or reagents whose preparation is not described explicitlyhereinafter were purchased commercially from generally accessiblesources. For all other reactants or reagents whose preparation likewiseis not described hereinafter and which were not commercially obtainableor were obtained from sources which are not generally accessible, areference is given to the published literature in which theirpreparation is described.

Starting Materials and Intermediates:

Intermediate C52

(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrol-2-yl]-2,2-dimethy1propan-1-amine

10.00 g (49.01 mmol) of methyl 4-bromo-1H-pyrrole-2-carboxylate wereinitially charged in 100.0 ml of DMF, and 20.76 g (63.72 mmol) ofcaesium carbonate and 9.22 g (53.91 mmol) of benzyl bromide were added.The reaction mixture was stirred at RT overnight. The reaction mixturewas partitioned between water and ethyl acetate and the aqueous phasewas extracted with ethyl acetate. The combined organic phases were driedover magnesium sulphate and the solvent was evaporated under reducedpressure. The reaction was repreated with 90.0 g of methyl4-bromo-1H-pyrrole-2-carboxylate. The two combined reactions werepurified by preparative RP-HPLC (column: Daiso 300×100; 10μ, flow rate:250 ml/min, MeCN/water). The solvents were evaporated under reducedpressure and the residue was dried under high vacuum. This gave 125.15 g(87% of theory) of the compound methyl1-benzyl-4-bromo-1H-pyrrole-2-carboxy late.

LC-MS (Method 1): R_(t)=1.18 min; MS (ESIpos): m/z=295 [M+H]⁺.

Under argon, 4.80 g (16.32 mmol) of methyl1-benzyl-4-bromo-1H-pyrrole-2-carboxylate were initially charged in DMF,and 3.61 g (22.85 mmol) of (2,5-difluorophenyl)boronic acid, 19.20 ml ofsaturated sodium carbonate solution and 1.33 g (1.63 mmol) of[1,1′-bis(diphenylphosphino)ferrocene]-dichloropalladium(II):dichloromethanewere added. The reaction mixture was stirred at 85° C. overnight. Thereaction mixture was filtered through Celite and the filter cake waswashed with ethyl acetate. The organic phase was extracted with waterand then washed with saturated NaCl solution. The organic phase wasdried over magnesium sulphate and the solvent was evaporated underreduced pressure. The residue was purified by silica gel chromatography(mobile phase: cyclohexane/ethyl acetate 100:3). The solvents wereevaporated under reduced pressure and the residue was dried under highvacuum. This gave 3.60 g (67% of theory) of the compound methyl1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate.

LC-MS (Method 7): R_(t)=1.59 min; MS (ESIpos): m/z=328 [M+H]⁺.

3.60 g (11.00 mmol) of methyl1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carboxylate were initiallycharged in 90.0 ml of THF, and 1.04 g (27.50 mmol) of lithium aluminiumhydride (2.4 M in THF) were added at 0° C. The reaction mixture wasstirred at 0° C. for 30 minutes. At 0° C., saturated potassium sodiumtartrate solution was added, and ethyl acetate was added to the reactionmixture. The organic phase was extracted three times with saturatedpotassium sodium tartrate solution. The organic phase was washed oncewith saturated NaCl solution and dried over magnesium sulphate. Thesolvent was evaporated under reduced pressure and the residue wasdissolved in 30.0 ml of dichloromethane. 3.38 g (32.99 mmol) ofmanganese(IV) oxide were added, and the mixture was stirred at RT for 48h. Another 2.20 g (21.47 mmol) of manganese(IV) oxide were added, andthe mixture was stirred at RT overnight. The reaction mixture wasfiltered through Celite and the filter cake was washed withdichloromethane. The solvent was evaporated under reduced pressure andthe residue 2.80 g of(1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde) was usedwithout further purification in the next step of the synthesis.

LC-MS (Method 7): R_(t)=1.48 min; MS (ESIpos): m/z=298 [M+H]⁺.

28.21 g (94.88 mmol) of1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrole-2-carbaldehyde together with23.00 g (189.77 mmol) of (R)-2-methylpropane-2-sulphinamide wereinitially charged in 403.0 ml of absolute THF, and 67.42 g (237.21 mmol)of titanium(IV) isopropoxide were added and the mixture was stirred atRT overnight. 500.0 ml of saturated NaCl solution and 1000.0 ml of ethylacetate were added, and the mixture was stirred at RT for 1 h. Themixture was filtered through kieselguhr and the filtrate was washedtwice with saturated NaCl solution. The organic phase was dried overmagnesium sulphate, the solvent was evaporated under reduced pressureand the residue was purified using Biotage Isolera (silica gel, column1500+340 g SNAP, flow rate 200 ml/min, ethyl acetate/cyclohexane 1:10).

LC-MS (Method 7): R_(t)=1.63 min; MS (ESIpos): m/z=401 [M+H]⁺.

25.00 g (62.42 mmol) of(R)-N-{(E/Z)-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]methylene}-2-methylpropane-2-sulphinamidewere initially charged in absolute THF under argon and cooled to −78° C.12.00 g (187.27 mmol) of tert-butyllithium (1.7 M solution in pentane)were then added at −78° C. and the mixture was stirred at thistemperature for 3 h. At −78° C., 71.4 ml of methanol and 214.3 ml ofsaturated ammonium chloride solution were then added in succession, andthe reaction mixture was allowed to warm to RT and stirred at RT for 1h. The mixture was diluted with ethyl acetate and washed with water. Theorganic phase was dried over magnesium sulphate and the solvent wasevaporated under reduced pressure. The residue(R)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulphinamidewas used without further purification in the next step of the synthesis.

LC-MS (Method 6): R_(t)=2.97 min; MS (ESIpos): m/z=459 [M+H]⁺.

28.00 g (61.05 mmol) of(R)-N-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2-methylpropane-2-sulphinamidewere initially charged in 186.7 ml of 1,4-dioxane, and 45.8 ml of HCl in1,4-dioxane solution (4.0 M) were then added. The reaction mixture wasstirred at RT for 2 h and the solvent was evaporated under reducedpressure. The residue was purified by preparative RP-HPLC (column:Kinetix 100×30; flow rate: 60 ml/min, MeCN/water). The acetonitrile wasevaporated under reduced pressure and dichloromethane was added to theaqueous residue. The organic phase was washed with sodium bicarbonatesolution and dried over magnesium sulphate. The solvent was evaporatedunder reduced pressure and the residue was dried under high vacuum. Thisgave 16.2 g (75% of theory) of the title compound.

LC-MS (Method 6): R_(t)=2.10 min; MS (ESIpos): m/z=338 [M-NH₂]⁺, 709[2M+H]⁺.

¹H-NMR (400 MHz, DMSO-d 6): 6 [ppm]=0.87 (s, 9H), 1.53 (s, 2H), 3.59 (s,1H), 5.24 (d, 2H), 6.56 (s, 1H), 6.94 (m, 1H), 7.10 (d, 2H), 7.20 (m,1H), 7.26 (m, 2H), 7.34 (m, 2H), 7.46 (m, 1H).

Intermediate C58

(2S)-44{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-2-{[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoicacid

4.3 g (12.2 mmol) of Intermediate C52 were dissolved in 525 ml of DCM,and 3.63 g (17.12 mmol) of sodium triacetoxyborohydride and 8.4 ml ofacetic acid were added. After 5 min of stirring at RT, 8.99 g (24.5mmol) of Intermediate L57 dissolved in 175 ml of DCM were added and thereaction was stirred at RT for a further 45 min. The reaction was thendiluted with 300 ml of DCM and washed twice with 100 ml of sodiumbicarbonate solution and once with saturated NaCl solution. The organicphase was dried over magnesium sulphate, the solvent was evaporatedunder reduced pressure and the residue was dried under high vacuum. Theresidue was then purified by preparative RP-HPLC (column: ChromatorexC18). After combination of the appropriate fractions, the solvent wasevaporated under reduced pressure and the residue was dried under highvacuum. This gave 4.6 g (61% of theory) of methyl(2S)-4-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate.

LC-MS (Method 12): R_(t)=1.97 min; MS (ESIpos): m/z=614 (M+H)⁺.

2.06 g (3.36 mmol) of this intermediate were initially charged in 76 mlof DCM and acylated with 0.81 ml (7.17 mmol) of 2-chloro-2-oxoethylacetate in the presence of 2.1 ml of triethylamine. After 20 h ofstirring at RT, 0.36 ml of 2-chlor-2-oxoethyl acetate and 0.94 ml oftriethylamine were added and the reaction was stirred at RT for afurther 15 min. The mixture was then diluted with 500 ml of ethylacetate and extracted successively twice with 300 ml of 5% strengthcitric acid, twice with 300 ml of saturated sodium bicarbonate solutionand once with 100 ml of saturated sodium chloride solution and thendried over magnesium sulphate and concentrated. Drying under high vacuumgave 2.17 g (79% of theory) of the protected intermediate.

LC-MS (Method 1): R_(t)=1.48 min; MS (ESIpos): m/z=714 (M+H)⁺.

2.17 mg (2.64 mmol) of this intermediate were dissolved in 54 ml of THFand 27 ml of water, and 26 ml of a 2-molar lithium hydroxide solutionwere added. The mixture was stirred at RT for 30 min and then adjustedto a pH between 3 and 4 using 1.4 ml of TFA. The mixture wasconcentrated under reduced pressure. Once most of the THF had beendistilled off, the aqueous solution was extracted twice with DCM andthen concentrated to dryness under reduced pressure. The residue waspurified by preparative HPLC (column: Chromatorex C18). Aftercombination of the appropriate fractions, the solvent was evaporatedunder reduced pressure and the residue was lyophilized fromacetonitrile/water. This gave 1.1 g (63% of theory) of the titlecompound.

LC-MS (Method 1): R_(t)=1.34 min; MS (ESIpos): m/z=656 (M−H)⁻.

¹H-NMR (400 MHz, DMSO-d₆): δ [ppm]=0.03 (s, 9H), 0.58 (m, 1H), 0.74-0.92(m, 11H), 1.40 (m, 1H), 3.3 (m, 2H), 3.7 (m, 1H), 3.8-4.0 (m, 2H), 4.15(q, 2H), 4.9 and 5.2 (2d, 2H), 5.61 (s, 1H), 6.94 (m, 2H), 7.13-7.38 (m,7H), 7.48 (s, 1H), 7.60 (m, 1H), 12.35 (s, 1H).

Intermediate C70

(2-(Trimethylsilyl)ethyl{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate

990.0 mg (2.79 mmol) of(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropan-1-amine(intermediate C52) were initially charged in 15.0 ml of dichloromethane,and 828.8 mg (3.91 mmol) of sodium triacetoxyborohydride and 129.9 mg(3.21 mmol) of acetic acid were added, and the mixture was stirred at RTfor 5 min. 698.1 mg (3.21 mmol) of 2-(trimethylsilyl)ethyl(3-oxopropyl)carbamate (Intermediate L58) dissolved in 15.0 ml ofdichloromethane were added, and the reaction mixture was stirred at RTovernight. The reaction mixture was diluted with ethyl acetate and theorganic phase was washed in each case twice with saturated sodiumcarbonate solution and saturated NaCl solution. The organic phase wasdried over magnesium sulphate and the solvent was evaporated underreduced pressure. The residue was purified on silica gel (mobile phase:dichloromethane/methanol=100:2). The solvents were evaporated underreduced pressure and the residue was dried under high vacuum. This gave1.25 g (73% of theory) of the compound 2-(trimethylsilyl)ethyl[3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamate.

LC-MS (Method 1): R_(t)=1.09 min; MS (ESIpos): m/z=556 (M+H)⁺.

908.1 mg (1.63 mmol) of 2-(trimethylsilyl)ethyl[3-({(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino)propyl]carbamateand 545.6 mg (5.39 mmol) of triethylamine were initially charged in 10.0ml of dichloromethane, and the mixture was cooled to 0° C. At thistemperature, 590.5 mg (5.23 mmol) of chloroacetyl chloride were addedand the mixture was stirred at RT overnight. The reaction mixture wasdiluted with ethyl acetate and the organic phase was washed in each casethree times with saturated sodium bicarbonate solution and saturatedammonium chloride solution. The organic phase was washed with saturatedNaCl solution and dried over magnesium sulphate. The residue waspurified by preparative RP-HPLC (column: Reprosil 250×30; 10μ, flowrate: 50 ml/min, MeCN/water, 0.1% TFA). The solvents were evaporatedunder reduced pressure and the residue was dried under high vacuum. Thisgave 673.8 mg (65% of theory) of the title compound.

LC-MS (Method 1): R_(t)=1.53 min; MS (ESIneg): m/z=676 (M+HCOO⁻)⁻.

Intermediate C71

S-(11-{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-L-cysteine/trifluoroaceticacid (1:1)

536.6 mg (4.43 mmol) of L-cysteine were suspended in 2.5 ml of watertogether with 531.5 mg (6.33 mmol) of sodium bicarbonate. 400.0 mg (0.63mmol) of 2-(trimethylsilyl)ethyl{3-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(chloroacetyl)amino]propyl}carbamate(Intermediate C70) dissolved in 25.0 ml of isopropanol and 1.16 g (7.59mmol) of 1,8-diazabicyclo[5.4.0]undec-7-ene were added. The reactionmixture was stirred at 50° C. for 1.5 h. Ethyl acetate was added to thereaction mixture and the organic phase was washed repeatedly withsaturated sodium bicarbonate solution and once with sat. NaCl solution.The organic phase was dried over magnesium sulphate, the solvent wasevaporated under reduced pressure and the residue was dried under highvacuum. The residue was purified by preparative RP-HPLC (column:Reprosil 250×30; 10μ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). Thesolvents were evaporated under reduced pressure and the residue wasdried under high vacuum. This gave 449.5 mg (86% of theory) of the titlecompound.

LC-MS (Method 1): R_(t)=1.20 min; MS (ESIpos): m/z=717 (M+H)⁺.

Intermediate C80

S-(11-{(1R)-1-[1-Benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-N-[15-(glycylamino)-4,7,10,13-tetraoxapentadecan-1-oyl]-L-cysteinetrifluoroacetic acid (1:1)

Under argon, 43.4 mg (95.1 μmol) of1-({N-[(benzyloxy)carbonyl]glycyl}amino)-3,6,9,12-tetraoxapentadecan-15-oicacid (Intermediate L90) were initially charged in 2.5 ml of DMF, 14.6 mg(95.1 μmol) of 1-hydroxy-1H-benzotriazole hydrate, 30.5 mg (95.1 μmol)of (benzotriazol-1-yloxy)bisdimethylaminomethylium fluoroborate and 16.5μl (95.1 μmol) of N,N-diisopropylethylamine were added and the mixturewas stirred for 10 min. 79.0 mg (95.1 μmol) ofS-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-L-cysteinetrifluoroacetic acid (1:1) (Intermediate C71) were dissolved in 2.5 mlof DMF, 49.5 μl (285.3 μmol) of N,N-diisopropylethylamine were added andthe mixture was added to the reaction. The reaction mixture was stirredat RT for 2 h and purified directly by preparative RP-HPLC (column:Reprosil 125×30; 10μ, flow rate: 50 ml/min, MeCN/water, 0.1% TFA). Thesolvents were evaporated under reduced pressure and the residue wasdried under high vacuum. This gave 44.2 mg (40% of theory) of thecompoundS-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-N-[15-({N-[(benzyloxy)carbonyl]glycyl}amino)-4,7,10,13-tetraoxapentadecan-1-oyl]-L-cysteine.

LC-MS (Method 12): R_(t)=2.57 min; MS (ESIpos): m/z=1156 [M+H]⁺

60.2 mg (52.1 μmol) ofS-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-N-[15-({N-[(benzyloxy)carbonyl]glycyl}amino)-4,7,10,13-tetraoxapentadecan-1-oyl]-L-cysteinewere suspended in 3.0 ml of ethanol, 6.0 mg of palladium on activatedcarbon (10%) were added and the mixture was hydrogenated with hydrogenat RT and standard pressure for 1 h. Twice, 6.0 mg of palladium onactivated carbon (10%) were added and the mixture was hydrogenated withhydrogen at RT and standard pressure for 1 h. The catalyst was filteredoff and the reaction mixture was freed from the solvent under reducedpressure and dried under high vacuum. The residue was purified bypreparative RP-HPLC (column: Reprosil 125×30; 10μ, flow rate: 50 ml/min,MeCN/water, 0.1% TFA). The solvents were evaporated under reducedpressure and the residue was dried under high vacuum. This gave 29.4 mg(50% of theory) of the title compound.

LC-MS (Method 5): R_(t)=3.77 min; MS (ESIpos): m/z=1021 [M+H]⁺

Intermediate L1

Trifluoroaceticacid/N-(2-aminoethyl)-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetamide(1:1)

The title compound was prepared by classical methods of peptidechemistry from commercially available(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetic acid and tert-butyl(2-aminoethyl)carbamate.

HPLC (Method 11): R_(t)=0.19 min;

LC-MS (Method 1): R_(t)=0.17 min; MS (ESIpos): m/z=198 (M+H)⁺.

Intermediate L57

Methyl (2S)-4-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoate

500.0 mg (2.72 mmol) of methyl L-asparaginate hydrochloride and 706.3 mg(2.72 mmol) of 2-(trimethylsilyl)ethyl2,5-dioxopyrrolidine-1-carboxylate were initially charged in 5.0 ml of1,4-dioxane, and 826.8 mg (8.17 mmol) of triethylamine were added. Thereaction mixture was stirred at RT overnight. The reaction mixture waspurified directly by preparative RP-HPLC (column: Reprosil 250×40; 10μ,flow rate: 50 ml/min, MeCN/water, 0.1% TFA). The solvents were thenevaporated under reduced pressure and the residue was dried under highvacuum. This gave 583.9 mg (74% of theory) of the compound(3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoicacid.

LC-MS (Method 1): R_(t)=0.89 min; MS (ESIneg): m/z=290 (M−H)⁻.

592.9 mg of(3S)-4-methoxy-4-oxo-3-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)butanoicacid were initially charged in 10.0 ml of 1,2-dimethoxyethane, themixture was cooled to −15° C. and 205.8 mg (2.04 mmol) of4-methylmorpholine and 277.9 mg (2.04 mmol) of isobutyl chloroformatewere added. The precipitate was filtered off with suction after 15 minand twice with in each case 10.0 ml of 1,2-dimethoxyethane. The filtratewas cooled to −10° C., and 115.5 mg (3.05 mmol) of sodium borohydridedissolved in 10 ml of water were added with vigorous stirring. Thephases were separated and the organic phase was washed in each case oncewith saturated sodium bicarbonate solution and saturated NaCl solution.The organic phase was dried over magnesium sulphate, the solvent wasevaporated under reduced pressure and the residue was dried under highvacuum. This gave 515.9 mg (91% of theory) of the compound methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserinate.

LC-MS (Method 1): R_(t)=0.87 min; MS (ESIpos): m/z=278 (M+H)⁺.

554.9 mg (2.00 mmol) of methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-homoserinate were initiallycharged in 30.0 ml of dichloromethane, and 1.27 g (3.0 mmol) ofDess-Martin periodinane and 474.7 mg (6.00 mmol) of pyridine were added.The mixture was stirred at RT overnight. After 4 h, the reaction wasdiluted with dichloromethane and the organic phase was washed in eachcase three times with 10% strength Na₂S₂O₃ solution, 10% strength citricacid solution and saturated sodium bicarbonate solution.

The organic phase was dried over magnesium sulphate and the solvent wasevaporated under reduced pressure. This gave 565.7 mg (97% of theory) ofthe title compound.

¹H-NMR (400 MHz, DMSO-d₆): δ [ppm]=0.03 (s, 9H), 0.91 (m, 2H), 2.70-2.79(m, 1H), 2.88 (dd, 1H), 3.63 (s, 3H), 4.04 (m, 2H), 4.55 (m, 1H), 7.54(d, 1H), 9.60 (t, 1H).

Intermediate L58

2-(Trimethylsilyl)ethyl (3-oxopropyl)carbamate

434.4 mg (5.78 mmol) of 3-amino-1-propanol and 1.50 g (5.78 mmol) of2-(trimethylsilyl)ethyl 2,5-dioxopyrrolidine-1-carboxylate weredissolved in 10.0 ml of dichloromethane, 585.3 mg (5.78 mmol) oftriethylamine were added and the mixture was stirred at RT overnight.The reaction mixture was diluted with dichloromethane and the organicphase was washed with water and saturated sodium bicarbonate solutionand then dried over magnesium sulphate. The solvent was evaporated underreduced pressure. The residue 2-(trimethylsilyl)ethyl(3-hydroxypropyl)carbamate (996.4 mg, 79% of theory) was dried underhigh vacuum and used without further purification in the next step ofthe synthesis. 807.0 mg (3.68 mmol) of 2-(trimethylsilyl)ethyl(3-hydroxypropyl)carbamate were initially charged in 15.0 ml ofchloroform and 15.0 ml of 0.05 N potassium carbonate/0.05 N sodiumbicarbonate solution (1:1). 102.2 mg (0.37 mmol) oftetra-n-butylammonium chloride, 736.9 mg (5.52 mmol) ofN-chlorosuccinimide and 57.5 mg (0.37 mmol) of TEMPO were then added andthe reaction mixture was stirred vigorously at RT overnight. Thereaction mixture was diluted with dichloromethane and the organic phasewas washed with water and saturated NaCl solution. The organic phase wasdried over magnesium sulphate and the solvent was evaporated underreduced pressure. The residue was dried under high vacuum and usedwithout further purification in the next step of the synthesis (890.3mg).

Intermediate L74

3-[2-[2-[2-[2-[[2-(2,5-Dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoicacid

107 mg (0.335 mmol) of tert-butyl3-[2-[2-[2-(2-aminoethoxy)ethoxy]ethoxy]ethoxy]propanoate and 93 mg(0.369 mmol) of (2,5-dioxopyrrolidin-1-yl)2-(2,5-dioxopyrrol-1-yl)acetate were dissolved in 5 ml ofdimethylformamide, and 0.074 ml (0.671 mmol) of N-methylmorpholine wereadded. The reaction mixture was stirred at RT overnight. 0.048 ml (0.838mmol) of acetic acid were added and the reaction mixture was purifieddirectly by preparative RP-HPLC (column: Reprosil 125×30; 10μ, flowrate: 50 ml/min, MeCN/water/0.1% TFA). The solvents were evaporatedunder reduced pressure and the residue was dried under high vacuum. Thisgave 133 mg (86%, purity 100%) of tert-butyl3-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate.

LC-MS (Method 1): R_(t)=0.82 min; MS (ESIpos): m/z=459 (M+H)⁺.

0.5 ml of TFA was added to a solution of tert-butyl3-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoate(130 mg, 0.284 mmol) in 5 ml of dichloromethane. The reaction mixturewas stirred at RT overnight. The reaction mixture was concentrated underreduced pressure and the residue was taken up in water and lyophilized.The residue was used further without further purification. This gave 102mg (90%, purity 100%) of the title compound.

LC-MS (Method 1): R_(t)=0.52 min; MS (ESIpos): m/z=402 (M+H)⁺.

Intermediate L90

1-({N-[(Benzyloxy)carbonyl]glycyl}amino)-3,6,9,12-tetraoxapentadecan-15-oicacid

118 mg (566 μmol) of N-[(benzyloxy)carbonyl]glycine were initiallycharged in 5.0 ml of DMF, 200 mg (622 μmol) of tert-butyl1-amino-3,6,9,12-tetraoxapentadecan-15-oate, 130 mg (849 μmol) of1-hydroxy-1H-benzotriazole hydrate and 130 mg (679 μmol) of1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride were addedand the mixture was stirred at RT for 1 h. Ethyl acetate was added andthe mixture was extracted twice with 5% strength citric acid solutionand with saturated sodium bicarbonate solution. The organic phase waswashed twice with saturated sodium chloride solution and dried overmagnesium sulphate. The solvents were evaporated under reduced pressureand the residue was dried under high vacuum. This gave 274 mg (95% oftheory) of tert-butyl1-({N-[(benzyloxy)carbonyl]glycyl}amino)-3,6,9,12-tetraoxapentadecan-15-oate.

LC-MS (Method 12): Rt=1.69 min; MS (ESIpos): m/z=513 (M+H)⁺.

820 μl (11 mmol) of TFA were added to a solution of 274 mg (535 μmol) oftert-butyl1-({N-[(benzyloxy)carbonyl]glycyl}amino)-3,6,9,12-tetraoxapentadecan-15-oatein 5.0 ml of dichloromethane. The reaction mixture was stirred at RT for3 h. The reaction mixture was concentrated under reduced pressure andthe residue was taken up in water and lyophilized. This gave 262 mg(100% of theory) of the title compound.

LC-MS (Method 12): R_(t)=1.12 min; MS (ESIpos): m/z=457 (M+H)⁺.

Intermediate F104

Trifluoroaceticacid/(2S)-2-amino-4-[{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}(glycoloyl)amino]-N-(2-{[(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)acetyl]amino}ethyl)butanamide(1:1)

15 mg (0.023 mmol) of Intermediate C58 were initally reacted with 11 mg(0.036 mmol) of Intermediate L1 in the presence of 13 mg (0.034 mmol) ofHATU and 10 μl of N,N-diisopropylethylamine. After 60 min of stirring atRT, the mixture was concentrated and the residue was purified bypreparative HPLC. This gave 12.3 mg (63% of theory) of the protectedintermediate.

LC-MS (Method 1): R_(t)=1.3 min; MS (EIpos): m/z=837 [M+H]⁺.

In the second step, this intermediate was dissolved in 3 ml of2,2,2-trifluoroethanol. 12 mg (0.088 mmol) of zinc chloride were added,and the reaction was stirred at 50° C. for 2 h. 26 mg (0.088 mmol) ofethylenediamine-N,N,N′,N′-tetraacetic acid and 2 ml of a 0.1% strengthaqueous trifluoroacetic acid solution were then added. The reaction waspurified by preparative HPLC. Concentration of the appropriate fractionsand lyophilization of the residue from acetonitrile/water gave 8.1 mg(68% of theory) of the title compound.

LC-MS (Method 1): R_(t)=0.89 min; MS (ESIpos): m/z=693 (M+H)⁺.

Intermediate F257

R-{2-[(3-Aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}-N-[18-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azaoctadecan-1-oyl]-L-cysteine/trifluoroaceticacid (1:1)

50.0 mg (0.06 mmol) ofR-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-L-cysteine/trifluoroaceticacid (1:1) (Intermediate C71) and 29 mg (0.07 mmol) of3-[2-[2-[2-[2-[[2-(2,5-dioxopyrrol-1-yl)acetyl]amino]ethoxy]ethoxy]ethoxy]ethoxy]propanoicacid (Intermediate L74) were dissolved in 3.0 ml of DMF, and 27.3 mg(0.07 mmol) of HATU and 23.3 mg (0.18 mmol) of N,N-diisopropylethylaminewere added. The reaction mixture was stirred at RT for 2 hours. Thereaction mixture was purified directly by preparative RP-HPLC (column:Reprosil 125×30; 10μ, flow rate: 50 ml/min, MeCN/water/0.1% TFA). Thesolvents were evaporated under reduced pressure and the residue wasdried under high vacuum. This gave 17.4 mg (26%) of the compoundR-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-N-[18-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azaoctadecan-1-oyl]-L-cysteine.

LC-MS (Method 6): R_(t)=1.34 min; MS (ESIpos): m/z=1101 (M+H)⁺.

17 mg (0.02 mmol) ofR-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-N-[18-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)-17-oxo-4,7,10,13-tetraoxa-16-azaoctadecan-1-oyl]-L-cysteinewere dissolved in 1.0 ml of trifluoroethanol, and 6.3 mg (0.05 mmol) ofzinc dichloride were added. The reaction mixture was stirred at 50° C.overnight. 13.5 mg (0.05 mmol) of ethylenediamine-N,N,N′,N′-tetraaceticacid were added, the reaction mixture was stirred for 10 min and water(0.1% TFA) was then added. Purification was carried out directly bypreparative RP-HPLC (column: Reprosil 125×30; 10μ, flow rate: 50 ml/min,MeCN/water, 0.1% TFA). The solvents were evaporated under reducedpressure and the residue was dried under high vacuum. This gave 7.6 mg(46%) of the title compound.

LC-MS (Method 1): R_(t)=0.91 min; MS (ESIpos): m/z=957 (M+H)⁺.

B: Preparation of Antibody Drug Conjugates (ADC)

B-1. General Process for Generating Anti-CD123 Antibodies

The anti-CD123 antibodies were obtained by CDR grafting. The sequence ofthe 7G3 antibody (EP2426148) represents the starting point of thehumanized antibodies such as TPP-5969, TPP-8987 and TPP-9476. BispecificscFv immunofusion proteins based on 12F1 are disclosed in WO2013/173820.

Based on the publication of the sequences of the variable regions (VHand VL) of 12F1 (WO 2013/173820), the following antibody sequences wereobtained by fusion of the variable domains of the donor immunoglobulin(VH and VL) with the constant regions of a human antibody. A chimericvariant of 12F1 was generated. Humanized anti-CD123 antibodies wereobtained by CDR grating representing the starting point of the humanizedantibodies such as TPP-8988 and TPP-9342.

Humanization

The murine antibody sequence of the 7G3 antibody was humanized bytransferring the CDRs into a human antibody skeleton. For the definitionof the CDRs according to Kabat, see Andre C. R. Martin, “Proteinsequence and structure analysis of antibody variable domains” inAntibody Engineering (Springer Lab Manuals), Eds.: Duebel, S. andKontermann, R., Springer-Verlag, Heidelberg. After comparison of themurine frame sequences (without CDRs) with human germline sequences, asimilar frequently occuring human frame sequence was selected. In thiscase, it was the heavy chain IGHV1-46-01 with the J sequence IGHJ4-03and the light chain IGKV4-1-01 with the J segment IGKJ2. The germlinesequences originated from the VBASE2 database (Retter I, Althaus HH,Münch R, MGller W: VBASE2, an integrative V gene database. Nucleic AcidsRes. 2005 Jan. 1; 33(Database issue):D671-4).

The sequences were named using the IMGT system (Lefranc, M.-P.,Giudicelli, V., Ginestoux, C., Jabado-Michaloud, J., Folch, G.,Bellahcene, F., Wu, Y., Gemrot, E., Brochet, X., Lane, J., Regnier, L.,Ehrenmann, F., Lefranc, G. and Duroux, P. IMGT®, the internationalImMunoGeneTics information system®. Nucl. Acids Res, 37, D1006-D1012(2009); doi:10.1093/nar/gkn838). The antibody variants TPP-5969,TPP-8987 and TPP-9476 described herein carry various point mutationsdiffering from the human germline sequence which may influnce theirproperties. Besides the chimeric antibody TPP-6013 humanized antibodieswere obtained by transferring the CDRs into a human antibody skeleton asTPP-8988 and TPP-9342

B-2. General Process for Expressing Anti-CD123 Antibodies in MammalianCells

The antibodies, for example TPP-6013, TPP-5969, TPP-8987, TPP-8988,TPP-9476 and TPP-9342 were produced in transient cultures of mammaliancells, as described by Tom et al., Chapter 12 in Methods Express:Expression Systems, edited by Micheal R. Dyson and Yves Durocher, ScionPublishing Ltd, 2007.

B-3. General Process for Purifying Antibodies from Cell Supernatants

The antibodies, for example TPP-8987 and TPP-8988, were obtained fromthe cell culture supernatants. The cell supernatants were clarified bycentrifugation of cells. The cell supernatant was then purified byaffinity chromatography on a MabSelect Sure (GE Healthcare)chromatography column. To this end, the column was equilibrated in DPBSpH 7.4 (Sigma/Aldrich), the cell supernatant was applied and the columnwas washed with about 10 column volumes of DPBS pH 7.4+500 mM sodiumchloride. The antibodies were eluted in 50 mM sodium acetate pH 3.5+500mM sodium chloride and then purified further by gel filtrationchromatography on a Superdex 200 column (GE Healthcare) in DPBS pH 7.4.

The antibodies were furthermore characterized by their binding affinityto soluble IL3Ralpha (obtained from R&D Systems) via BIAcore analysis.To determine the cell binding characteristics of the anti-CD123antibodies, binding was measured by flow cytometry on CD123-positivecancer cell line MOLM-13.

B-4. General Process for Coupling to Cysteine Side Chains

The following antibodies were used for the coupling reactions:

TPP-5969

TPP-6013

TPP-8987

TPP-8988

TPP-9476

The coupling reactions were usually carried out under argon.

Between 2 and 5 equivalents of tris(2-carboxyethyl)phosphinehydrochloride (TCEP), dissolved in PBS buffer, were added to a solutionof the appropriate antibody in PBS buffer in the concentration rangebetween 1 mg/ml and 20 mg/ml, preferably in the range of about 10 mg/mlto 15 mg/ml, and the mixture was stirred at RT for 30 min. For thispurpose, the solution of the respective antibody used can be employed atthe concentrations stated in the working examples, or it may optionallyalso be diluted with PBS buffer to about half of the stated startingconcentrations in order to get into the preferred concentration range.Subsequently, depending on the intended loading, from 2 to 12equivalents, preferably about 5-10 equivalents of the maleinimideprecursor compound to be coupled were added as a solution in DMSO. Here,the amount of DMSO should not exceed 10% of the total volume. Thereaction was stirred for 60-240 min at RT and then diluted with PBSbuffer which had been adjusted to pH 8 beforehand. The solution was thenapplied to PBS pH 8-equilibrated PD 10 columns (Sephadex® G-25, GEHealthcare) and eluted with PBS pH 8 buffer. The eluate may be dilutedwith PBS buffer pH 8. This solution was stirred at RT under argonovernight. The solution was then rebuffered to pH 7.2. The sample wasthen concentrated by ultracentrifugation and optionally rediluted withPBS buffer. If required, for better removal of low-molecular weightcomponents, concentration by ultrafiltration was repeated afterredilution with PBS buffer. For biological tests, if required, theconcentrations of the final ADC samples were optionally adjusted to therange of 0.5-15 mg/ml by redilution. The respective proteinconcentrations, stated in the working examples, of the ADC solutionswere determined. Furthermore, antibody loading (drug/mAb ratio) wasdetermined using the methods described under B-7.

The ADCs shown in the examples may also be present to a lesser or higherdegree in the form of the closed succinamides attached to theantibodies.

In the structural formulae shown, AK_(TPP-XXXX) or AK has the meaning

-   AK=anti-CD123 antibody (partially reduced)−S§ ¹-   AKTPP₅₉₆₉=anti-CD123 TPP-5969 (partially reduced)−S§ ¹-   AKTPP₆₀₁₃=anti-CD123 TPP-6013 (partially reduced)−S§ ¹-   AKTPP₈₉₈₇=anti-CD123 TPP-8987 (partially reduced)−S§ ¹-   AKTPP₈₉₈₈=anti-CD123 TPP-8988 (partially reduced)−S§ ¹-   AKTPP₉₄₇₆=anti-CD123 TPP-9476 (partially reduced)−S§ ¹-   where-   § ¹ represents the linkage to the succinimide group or to any    isomeric hydrolysed open-chain succinamides or the alkylene radical    resulting therefrom,-   and-   S represents the sulphur atom of a cysteine residue of the partially    reduced antibody.

B-6a. General Process for Preparing Isomeric Open Succinamide-CvsteineAdducts:

In an exemplary embodiment, 68 μmol of the maleinimide precursorcompounds described above were taken up in 15 ml of DMF, and with 36 mg(136 μmol) of N-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteine wereadded. The reaction mixture was stirred at RT for ˜20 h, thenconcentrated under reduced pressure and then purified by preparativeHPLC. The appropriate fractions were combined and the solvents wereevaporated under reduced pressure, and the residue was then dissolved in15 ml of THF/water 1:1. 131 μl of a 2M aqueous lithium hydroxidesolution were added and the reaction was stirred at RT for 1 h. Thereaction was then neutralized with a 1 M hydrochloric acid, the solventwas evaporated under reduced pressure and the residue was purified bypreparative HPLC. This gave ˜50% of theory of the regioisomericprotected intermediates as a colourless foam.

In the last step, 0.023 mmol of these regioisomeric hydrolysis productswere dissolved in 3 ml of 2,2,2-trifluoroethanol. 12.5 mg (0.092 mmol)of zinc chloride were added, and the reaction was stirred at 50° C. for4 h. 27 mg (0.092 mmol) of ethylenediamine-N,N,N′,N′-tetraacetic acidwere then added, and the solvent was evaporated under reduced pressure.The residue was purified by preparative HPLC. Concentration of theappropriate fractions and lyophilization of the residue fromacetonitrile/water gave the hydrolysed open sulphanylsuccinamides as aregioisomer mixture.

Further Purification and Characterization of the Conjugates According tothe Invention

After the reaction, in some instances the reaction mixture wasconcentrated, for example by ultrafiltration, and then desalted andpurified by chromatography, for example using a Sephadex® G-25 column.Elution was carried out, for example, with phosphate-buffered saline(PBS). The solution was then sterile filtered and frozen. Alternatively,the conjugate can be lyophylized.

B7. Determination of the Antibody, the Toxophor Loading and theProportion of Open Cysteine Adducts

For protein identification in addition to molecular weight determinationafter deglycosylation and/or denaturing, a tryptic digestion was carriedout which, after denaturing, reduction and derivatization, confirms theidentity of the protein via the tryptic peptides found.

The toxophor loading of the PBS buffer solutions obtained of theconjugates described in the working examples was determined as follows:Determination of toxophor loading of lysine-linked ADCs was carried outby mass spectrometric determination of the molecular weights of theindividual conjugate species. Here, the antibody conjugates were firstdeglycosylated with PNGaseF, and the sample was acidified and, afterHPLC separation/desalting, analysed by mass spectrometry usingESI-MicroTofQ (Bruker Daltonik). All spectra over the signal in the TIC(Total Ion Chromatogram) were added and the molecular weight of thedifferent conjugate species was calculated based on MaxEntdeconvolution. The DAR (=drug/antibody ratio) was then calculated aftersignal integration of the different species.

The toxophor loading of cysteine-linked conjugates was determined byreversed-phase chromatography of the reduced and denatured ADCs.Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution ofDL-dithiothreitol (DTT) (500 mM, 3 μl) were added to the ADC solution (1mg/ml, 50 μl). The mixture was incubated at 55° C. for one hour andanalysed by HPLC.

HPLC analysis was carried out on an Agilent 1260 HPLC system withdetection at 220 nm. A Polymer Laboratories PLRP-S polymericreversed-phase column (catalogue number PL1912-3802) (2.1×150 mm, 8 μmparticle size, 1000 A) was used at a flow rate of 1 ml/min with thefollowing gradient: 0 min, 25% B; 3 min, 25% B; 28 min, 50% B. Mobilephase A consisted of 0.05% trifluoroacetic acid (TFA) in water, mobilephase B of 0.05% trifluoroacetic acid in acetonitrile.

The detected peaks were assigned by retention time comparison with thelight chain (L0) and the heavy chain (H0) of the non-conjugatedantibody. Peaks detected exclusively in the conjugated sample wereassigned to the light chain with one toxophor (L1) and the heavy chainswith one, two and three toxophors (H1, H2, H3).

Average loading of the antibody with toxophors was calculated from thepeak areas determined by integration as double the sum of HC load and LCload, where LC load is calculated from the sum of the toxophornumber-average weighed integration results of all LC peaks divided bythe sum of the singly weighed integration results of all LC peaks, andwhere the HC-load is calculated from the sum of the toxophornumber-average weighed integration results of all HC peaks divided bythe sum of the singly weighed integration results of all HC peaks. Inindividual cases, it may not be possible to determine the toxophor loadaccurately owing to co-elutions of some peaks.

In the cases where light and heavy chains could not be separatedsufficiently by HPLC, determination of toxophor loading ofcysteine-linked conjugates was carried out by mass spectrometricdetermination of the molecular weights of the individual conjugatespecies at light and heavy chain.

Guanidinium hydrochloride (GuHCl) (28.6 mg) and a solution ofDL-dithiothreitol (DTT) (500 mM, 3 μl) were added to the ADC solution (1mg/ml, 50 μl). The mixture was incubated for one hour at 55° C. andanalysed by mass spectrometry after online desalting using ESI-MicroTofQ(Bruker Daltonik).

For the DAR determination, all spectra were added over the signal in theTIC (Total Ion Chromatogram), and the molecular weight of the differentconjugate species at light and heavy chain was calculated based onMaxEnt deconvolution. Average loading of the antibody with toxophors wascalculated from the peak areas determined by integration as double thesum of HC load and LC load, where LC load is calculated from the sum ofthe toxophor number-average weighed integration results of all LC peaksdivided by the sum of the singly weighed integration results of all LCpeaks, and where the HC-load is calculated from the sum of the toxophornumber-average weighed integration results of all HC peaks divided bythe sum of the singly weighed integration results of all HC peaks.

To determine the proportion of the open cysteine adduct, the molecularweight area ratio of closed to open cysteine adduct (molecular weightdelta 18 Dalton) of all singly conjugated light and heavy chain variantswas determined. The mean of all variants yielded the proportion of theopen cysteine adduct.

B-8. Checking the Antigen-Binding of the ADCs

The capability of the binder of binding to the target molecule waschecked after coupling had taken place. The person skilled in the art isfamiliar with multifarious methods which can be used for this purpose;for example, the affinity of the conjugate can be checked using ELISAtechnology or surface plasmon resonance analysis (BIAcore™ measurement).The conjugate concentration can be measured by the person skilled in theart using customary methods, for example for antibody conjugates byprotein determination. (see also Doronina et al.; Nature Biotechnol.2003; 21:778-784 and Polson et al., Blood 2007; 1102:616-623).

Metabolite Embodiments

Example M20

(1R,4R,27R,33R)-1-Amino-32-(3-aminopropyl)-33-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-34,34-dimethyl-6,9,25,31-tetraoxo-13,16,19,22-tetraoxa-3,29-dithia-7,10,26,32-tetraazapentatriacontane-1,4,27-tricarboxylicacid/trifluoroacetic acid (1:2)

First, methyl L-cysteinate hydrochloride (1:1) was converted with1-({[2-(trimethylsilyl)ethoxy]carbonyl}oxy)pyrrolidine-2,5-dione in DMFin the presence of N,N-diisopropylethylamine into methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteinate.

408 mg (1.93 mmol) of commercially available3-bromo-4-methoxy-4-oxobutanoic acid and 180 mg (0.644 mmol) of methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteinate were dissolved in 8ml of DMF, and 147 mg (0.97 mmol) of 1,8-diazabicyclo[5.4.0]undec-7-enewere added. After 18 h of stirring at RT, another 136 mg (0.64 mmol) of3-bromo-4-methoxy-4-oxobutanoic acid and 147 mg (0.97 mmol) of1,8-diazabicyclo[5.4.0]undec-7-ene were added, and the mixture wasstirred at RT for a further 12 h and then concentrated under reducedpressure. The residue was purified by preparative HPLC. Combination ofthe appropriate fractions and evaporation of the solvents under reducedpressure gave 151 mg (57% of theory) of4-methoxy-3-{[(2R)-3-methoxy-3-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]sulphanyl}-4-oxobutanoic acid.

LC-MS (Method 12): R_(t)=1.74 min; MS (ESIneg): m/z=408 (M−H)⁻.

3.66 mg (8.93 μmol) of4-methoxy-3-{[(2R)-3-methoxy-3-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]sulphanyl}-4-oxobutanoicacid were coupled in the presence of 3.66 mg (8.93 μmol) of HATU and 1.6μl (15 μmol) of 4-methylmorpholine with 13.0 mg (7.44 μmol) ofS-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-N-[15-(glycylamino)-4,7,10,13-tetraoxapentadecan-1-oyl]-L-cysteine/trifluoroaceticacid (1:1) (Intermediate C80), giving, after HPLC purification, 3.9 mg(37% of theory) of the fully protected intermediateS-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorphenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-N-[15-({N-[(8R,11R)-8,11-bis(methoxycarbonyl)-2,2-dimethyl-6,13-dioxo-5-oxa-10-thia-7-aza-2-silatridecan-13-yl]glycyl}amino)-4,7,10,13-tetraoxapentadecan-1-oyl]-L-cysteine.3.90 mg (2.76 μmol) of this intermediate were then stirred at RT with 35μl of a 2-molar lithium hydroxide solution in 1.0 ml of THF/water 3:1for 15 min, resulting in the cleavage of both methyl ester groups.Purification by HPLC gave 3.60 mg (94% of theory) of the dicarboxylicacid derivative.

LC-MS (Method 5): R_(t)=4.83 min; MS (ESIpos): m/z=1385 [M+H]⁺.

Finally, 3.6 mg (2.6 μmol) of this intermediate were completelydeprotected with zinc chloride in trifluoroethanol as described above.The residue was purified by preparative HPLC. Concentration of theappropriate fractions and lyophilization of the residue fromacetonitrile/water gave 1.92 mg (55% of theory) of the title compound.

LC-MS (Method 5): R_(t)=2.72 min; MS (ESIneg): m/z=1094 [M−H]⁻.

Example M21

(2R,24S,27R)-27-Amino-2-[({2-[(3-aminopropyl){(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}amino]-2-oxoethyl}sulphanyl)methyl]-24-(carboxymethyl)-4,20,23-trioxo-7,10,13,16-tetraoxa-25-thia-3,19,22-triazaoctacosane-1,28-dioicacid/trifluoroacetic acid (1:2)

742.8 mg (3.3 mmol) of commercially available2-bromo-4-ethoxy-4-oxobutanoic acid and 802 mg (2.87 mmol) of methylN-{[2-(trimethylsilyl)ethoxy]carbonyl}-L-cysteinate were dissolved in 32ml of DMF, and 655.4 mg (4.31 mmol) of1,8-diazabicyclo[5.4.0]undec-7-ene were added. After 20 h of stirring atRT, the reaction was concentrated under reduced pressure and the residuewas purified by preparative HPLC. Combination of the appropriatefractions and evaporation of the solvents under reduced pressure gave521 mg (43% of theory) of4-ethoxy-2-{[(2R)-3-methoxy-3-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]sulphanyl}-4-oxobutanoic acid.

LC-MS (Method 5): R_(t)=3.13 min; MS (ESIpos): m/z=424 (M+H)⁺.

4.36 mg (10.3 μmol) of4-ethoxy-2-{[(2R)-3-methoxy-3-oxo-2-({[2-(trimethylsilyl)ethoxy]carbonyl}amino)propyl]sulphanyl}-4-oxobutanoicacid were coupled in the presence of 3.92 mg (10.3 μmol) of HATU and 1.9μl (17 μmol) of 4-methylmorpholine with 15.0 mg (8.59 μmol) ofS-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorophenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-N-[15-(glycylamino)-4,7,10,13-tetraoxapentadecan-1-oyl]-L-cysteine/trifluoroaceticacid (1:1) (Intermediate C80), giving, after HPLC purification, 3.6 mg(26% of theory) of the fully protected intermediateS-(11-{(1R)-1-[1-benzyl-4-(2,5-difluorphenyl)-1H-pyrrol-2-yl]-2,2-dimethylpropyl}-2,2-dimethyl-6,12-dioxo-5-oxa-7,11-diaza-2-silatridecan-13-yl)-N-[15-({N-[(8R,11S)-11-(2-ethoxy-2-oxoethyl)-8-(methoxycarbonyl)-2,2-dimethyl-6,12-dioxo-5-oxa-10-thia-7-aza-2-siladodecan-12-yl]glycyl}amino)-4,7,10,13-tetraoxapentadecan-1-oyl]-L-cysteine.

6.20 mg (2.82 μmol) of this intermediate were then stirred at RT with 35μl of a 2-molar lithium hydroxide solution in 1.0 ml of THF/water 1:1for 15 min, resulting in the cleavage of both ester groups.Acidification and purification by HPLC gave 3.60 mg (92% of theory) ofthe dicarboxylic acid derivative.

LC-MS (Method 5): R_(t)=4.71 min; MS (ESIpos): m/z=1385 [M+H]⁺.

Finally, 3.60 mg (1.69 μmol) of this intermediate were completelydeprotected with zinc chloride in trifluoroethanol as described above.The residue was purified by preparative HPLC. Concentration of theappropriate fractions and lyophilization of the residue fromacetonitrile/water gave 0.88 mg (39% of theory) of the title compound.

LC-MS (Method 5): R_(t)=2.72 min; MS (ESIneg): m/z=1094 [M−H]⁻.

Working Examples ADCs

The ADCs shown in the structural formulae of the Working examples, whichwere coupled to the cystein side chains of the antibodies via maleimideradicals, are, depending on the linker and the coupling procedure,mainly present in the ring-opened forms shown in each case. However, thepreparation(s) may comprise a small proportion of the ring-closed form.

The ring-closed form is:

Ref 208m1

Under argon, a solution of 1.4 mg of TCEP in 0.83 ml of PBS buffer wasadded to 250 mg of Anti-CD123 TPP-5969 in 21.7 ml of PBS (c=11.5 mg/ml).The reaction was stirred at RT for 30 min, and 10.76 mg (0.0133 mmol) ofIntermediate F104 dissolved in 2500 μl of DMSO were then added. After afurther 90 min of stirring at RT, the reaction was re-buffered to pH 8using PD-10 columns (Sephadex® G-25, GE Healthcare). The eluate wasstirred under argon at RT overnight and then re-buffered to pH 7.2 usingPD-10 columns. The eluate was then diluted to 125 ml with PBS buffer (pH7.2), concentrated by ultracentrifugation, rediluted with PBS buffer (pH7.2) and reconcentrated again. The ADC batch obtained was characterizedas follows:

Protein concentration: 9.79 mg/ml

Drug/mAb ratio: 2.9

Ref 208m3

Under argon, a solution of 1.4 mg of TCEP in 0.83 ml of PBS buffer wasadded to 250 mg of Anti-CD123 TPP-6013 in 21.7 ml of PBS (c=11.5 mg/ml).The reaction was stirred at RT for 30 min, and 10.76 mg (0.0133 mmol) ofIntermediate F104 dissolved in 2500 μl of DMSO were then added. After afurther 90 min of stirring at RT, the reaction was re-buffered to pH 8using PD-10 columns (Sephadex© G-25, GE Healthcare). The eluate wasstirred under argon at RT overnight and then re-buffered to pH 7.2 usingPD-10 columns. The eluate was then diluted to 125 ml with PBS buffer (pH7.2), concentrated by ultracentrifugation, rediluted with PBS buffer (pH7.2) and reconcentrated again. The ADC batch obtained was characterizedas follows:

Protein concentration: 11.06 mg/ml

Drug/mAb ratio: 3.4

Ref 257m1

Under argon, a solution of 1.4 mg of TCEP in 0.83 ml of PBS buffer wasadded to 250 mg of Anti-CD123 TPP-5969 in 21.7 ml of PBS (c=11.5 mg/ml).The reaction was stirred at RT for 30 min, and 8.92 mg (0.0083 mmol) ofIntermediate F257 dissolved in 2500 μl of DMSO were then added. After afurther 90 min of stirring at RT, the reaction was re-buffered to pH 8using PD-10 columns (Sephadex® G-25, GE Healthcare). The eluate wasstirred under argon at RT overnight and then re-buffered to pH 7.2 usingPD-10 columns. The eluate was then diluted to 125 ml with PBS buffer (pH7.2), concentrated by ultracentrifugation, rediluted with PBS buffer (pH7.2) and reconcentrated again. The ADC batch obtained was characterizedas follows:

Protein concentration: 11.27 mg/ml

Drug/mAb ratio: 3.6

Ref 257m3

Under argon, a solution of 1.4 mg of TCEP in 0.83 ml of PBS buffer wasadded to 250 mg of Anti-CD123 TPP-6013 in 21.7 ml of PBS (c=11.5 mg/ml).The reaction was stirred at RT for 30 min, and 8.92 mg (0.0083 mmol) ofIntermediate F257 dissolved in 2500 μl of DMSO were then added. After afurther 90 min of stirring at RT, the reaction was re-buffered to pH 8using PD-10 columns (Sephadex® G-25, GE Healthcare). The eluate wasstirred under argon at RT overnight and then re-buffered to pH 7.2 usingPD-10 columns. The eluate was then diluted to 125 ml with PBS buffer (pH7.2), concentrated by ultracentrifugation, rediluted with PBS buffer (pH7.2) and reconcentrated again. The ADC batch obtained was characterizedas follows:

Protein concentration: 13.36 mg/ml

Drug/mAb ratio: 3.3

Example 1a

Under argon, a solution of 0.057 mg of TCEP in 100 μl of PBS buffer wasadded to 10 mg of anti-CD123 TPP-8987 in 900 μl of PBS (c=11.11 mg/ml).The reaction was stirred at RT for 30 min. 0.357 mg (0.00033 mmol) ofIntermediate F257 dissolved in 100 μl of DMSO were then added. After afurther 90 min of stirring at RT, the reaction was re-buffered to pH 8using PD-10 columns (Sephadex® G-25, GE Healthcare). The eluate wasstirred under argon at RT overnight and then re-buffered to pH 7.2 usingPD-10 columns. The eluate was then diluted to 14 ml with PBS buffer (pH7.2), concentrated by ultracentrifugation, rediluted with PBS buffer (pH7.2) and reconcentrated again. The ADC batch obtained was characterizedas follows:

Protein concentration: 6.30 mg/ml

Drug/mAb ratio: 3.1

Example 1b

Under argon, a solution of 0.143 mg of TCEP in 250 μl of PBS buffer wasadded to 25 mg of anti-CD123 TPP-8988 in 2500 μl of PBS (c=10.0 mg/ml).The reaction was stirred at RT for 30 min. 0.893 mg (0.00083 mmol) ofIntermediate F257 dissolved in 250 μl of DMSO were then added. After afurther 90 min of stirring at RT, the reaction was re-buffered to pH 8using PD-10 columns (Sephadex® G-25, GE Healthcare). The eluate wasstirred under argon at RT overnight and then re-buffered to pH 7.2 usingPD-10 columns. The eluate was then diluted to 14 ml with PBS buffer (pH7.2), concentrated by ultracentrifugation, rediluted with PBS buffer (pH7.2) and reconcentrated again. The ADC batch obtained was characterizedas follows:

Protein concentration: 10.43 mg/ml

Drug/mAb ratio: 4.3

Example 1c

Under argon, a solution of 0.172 mg of TCEP in 300 μl of PBS buffer wasadded to 30 mg of anti-CD123 TPP-9476 in 3000 μl of PBS (c=10.0 mg/ml).The reaction was stirred at RT for 30 min. 1.071 mg (0.001 mmol) ofIntermediate F257 dissolved in 300 μl of DMSO were then added. After afurther 90 min of stirring at RT, the reaction was re-buffered to pH 8using PD-10 columns (Sephadex® G-25, GE Healthcare). The eluate wasstirred under argon at RT overnight and then re-buffered to pH 7.2 usingPD-10 columns. The eluate was then diluted to 14 ml with PBS buffer (pH7.2), concentrated by ultracentrifugation, rediluted with PBS buffer (pH7.2) and reconcentrated again. The ADC batch obtained was characterizedas follows:

Protein concentration: 11.04 mg/ml

Drug/mAb ratio: 4.4

C: Assessment of Biological Efficacy

The biological activity of the compounds according to the invention canbe shown in the assays described below:

C-1a Determination of the Cytotoxic Effects of the ADCs Directed AgainstCD123

The analysis of the cytotoxic effects of the anti-CD123 ADCs and thecorresponding metabolites was carried out with various cell lines:

NCI-H292: human mucoepidermoid lung carcinoma cells, ATCC-CRL-1848,standard medium: RPMI 1640 (Biochrom; #FG1215, stab. glutamine)+10% FCS(Biochrom; #S0415).

MOLM-13: human acute monocytic leukaemia cells obtained from peripheralblood (AML-M5a), DSMZ, No. ACC 554, standard medium: RPMI 1640 (Gibco;#21875-059, stab. L-glutamine)+20% heat inactivated FCS (Gibco, No.10500-064); CD123-positive.

THP-1: human monocytic leukaemia cells obtained from peripheral blood,ATCC, NO. TIB-202, standard medium: RPMI 1640 (Gibco; #21875-059, stab.L-glutamine)+10% heat inactivated FCS (Gibco, No. 10500-064)+2.5 g ofglucose (20% glucose solution, Gibco, No. 19002); CD123-positive.

MV-4-11: human biphcnotypic B ayelimooocytic leukiaEcu, cells obtainedfrom peripheral blood, ATCC-CRL-9591, standard medium: IMDM(ATCC:30-2005), +10% heat inactivated FCS (Gibco, No. 10500-064);CD123-positive.

KG-1: human acute myeloid leukaemia cells obtained from bone marrow,DSMZ, No. ACC 14, standard medium: RPMI 1640 (Gibco; #21875-059, stab.L-glutamine)+10% heat inactivated FCS (Gibco, No. 10500-064)+2.5 g ofglucose (20% glucose solution, Gibco, No. 19002); CD123-positive.

NB4: human acute promyelocytic leukemia cells obtained from bone marrow,DSMZ, No. ACC 207, standard medium: RPMI 1640+GlutaMAX I (Invitrogen61870)+10% heat inactivated FCS (Gibco, No. 10500-064)+2.5 g of glucose(20% glucose solution, Gibco, No. 19002)+10 mM Hepes (Invitrogen15630)+1 mM Sodium Pyruvate (Invitrogen 11360); CD123-negative

The cells were cultivated by the standard method as stated by theAmerican Tissue Culture Collection (ATCC) or the Leibniz-InstitutDSMZ-Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH (DSMZ)for the cell lines in question.

MTT assay

The cells were cultivated according to the standard method using thegrowth media listed under C-1. The test is carried out by detaching thecells with a solution of Accutase in PBS (Biochrom AG #L2143; in case ofadherent cell), pelleting, resuspending in culture medium, counting andsowing into a 96-well culture plate with white bottom (Costar #3610)(NCI H292: 2500 cells/well; MOLM-13: 2000 cells/well; THP-1: 8000cells/well; NB4: 7000 cells/well; KG-1: 5000 cells/well, MV-4-11: 5000cells/well in a total volume of 100 μl). The cells were then incubatedin an incubator at 37° C. and 5% carbon dioxide. After 6 h or 48 h(adherent cells) the medium was replaced. The antibody drug conjugatesor the metabolites in 10 μl of culture medium in concentrations from10⁻⁵M to 10⁻¹³M were then pipetted to the cells (in triplicate), and theassay was then incubated in an incubator at 37° C. and 5% carbondioxide. After 96 h, the cell proliferation was detected using the MTTassay (ATCC, Manassas, Virginia, USA, catalogue No. 30-1010K). To thisend, the MTT reagent was incubated with the cells for 4 h, followed bylysis of the cells overnight by addition of the detergent. The dyeformed was detected at 570 nm (Infinite M1000 pro, Tecan). The measureddata were used to calculate the IC₅₀ of the growth inhibition using theDRC (dose response curve). The proliferation of cells which were nottreated with test substance but were otherwise identically treated wasdefined as the 100% figure.

Table 1 below lists the IC₅₀ values of representative working examplesfor different anti-CD123 antibodies from this assay:

TABLE 1 MOLM 13 THP-1 MV-4-11 NB4 IC₅₀ [M] IC₅₀ [M] IC₅₀ [M] IC₅₀ [M]Example MTT assay MTT assay MTT assay MTT assay 1a 6.86E−10 4.11E−097.19E−08 1b 1.67E−11 5.19E−09 1c 1.37E−10 9.33E−10 Ref 3.28E−09 1.03E−101.73E−07 208m1 Ref 1.21E−09 1.60E−09 3.00E−07 208m3 Ref 1.51E−095.96E−10 5.00E−07 257m1 Ref 3.89E−10 2.56E−10 6.28E−08 257m3

The activity data reported relate to the working examples described inthe present experimental section, with the drug/mAB ratios indicated.The values may possibly deviate for different drug/mAB ratios. The IC₅₀values are means of severalindependent experiments or individual values.The action of the CD123 antibody drug conjugates was selective for therespective isotype comprising the respective linker and toxophore.

C-1b Determination of the Inhibition of the Kinesin Spindle ProteinKSP/Eg5 by Selected Examples

The motor domain of the human kinesin spindle protein KSP/Eg5(tebu-bio/Cytoskeleton Inc, No. 027EG01-XL) was incubated in aconcentration of 10 nM with microtubuli (bovine or porcine,tebu-bio/Cytoskeleton Inc) stabilized with 50 μg/ml taxol (Sigma No.T7191-5MG) for 5 min at RT in 15 mM PIPES, pH 6.8 (5 mM MgCl₂ and 10 mMDTT, Sigma). The freshly prepared mixture was aliquoted into a 384 MTP(Greiner bio-one REF 781096). The inhibitors to be examined atconcentrations of 1.0×10-6 M to 1.0×10⁻¹³ M and ATP (final concentration500 μM, Sigma) were then added. Incubation was at RT for 2 h. ATPaseactivity was detected by detecting the inorganic phosphate formed usingmalachite green (Biomol). After addition of the reagent, the assay wasincubated at RT for 50 min prior to detection of the absorption at awavelength of 620 nm. The positive controls used were monastrol (Sigma,M8515-1 mg) and ispinesib (AdooQ Bioscience A10486). The individual dataof the dose-activity curve are eight-fold determinations. The IC₅₀values are means of two independent experiments. The 100% control wasthe sample which had not been treated with inhibitors.

Table 2 below lists the IC₅₀ values of representative working examplesfrom the assay described and the corresponding cytotoxicity data (MTTassay).

TABLE 2 NCI-H292 KSP assay IC₅₀ [M] Examples IC₅₀ [M] MTT assay M201.50E−09 1.49E−07 M21 2.31E−09

The activity data reported relate to the working examples described inthe present experimental section.

C-2a Internalisation Assay

Internalisation is a key process which enables specific and efficientprovision of the cytotoxic payload in antigen-expressing cancer cellsvia antibody drug conjugates (ADC). This process is monitored viafluorescent labelling of specific CD123 antibodies and an isotypecontrol antibody labelled with a pH-sensitive fluorophor. First, thefluorescent dye was conjugated to lysines of the antibody. Conjugationwas carried out using a two-fold molar excess of CypHer 5E mono NHSester (Batch 357392, GE Healthcare) at pH 8.3. After the coupling, thereaction mixture was purified by gel chromatography (Zeba Spin DesaltingColumns, 40K, Thermo Scientific, No. 87768; elution buffer: DULBECCO'SPBS, Sigma-Aldrich, No. D8537) to eliminate excess dye and to adjust thepH. The protein solution was concentrated using VIVASPIN 500 columns(Sartorius stedim biotec VS0131). The dye load of the antibody wasdetermined by spectrophotometric analysis (NanoDrop) and subsequentcalculation using the following equation D: P=A_(dye)ε_(protein):(A₂₈₀−0.16A_(dye))ε_(dye). The dye load of the CD123 antibodies examinedhere and the isotype control were of a comparable order. In cell bindingassays, it was confirmed that the conjugation did not lead to a changein the affinity of the antibodies. The antigen to be examined isexpressed by haematopoietic suspension cells, and the internalisation ofthe labelled CD123 antibodies was therefore examined in an FACS-basedinternalisation assay. To this end, cell lines with different expressionlevels of the receptor were selected and tested. The cells (5×10⁴/well)were sown in a 96-MTP in a total volume of 100 μl (Greiner bio-one,CELLSTAR, 650 180, U-bottom). After addition of the anti-CD123 antibodyto be examined in a final concentration of 10 μg/ml, the cell batcheswere incubated at 37° C. for different periods (1 h, 2 h, 6 h, intriplicate). The same treatment protocol was applied to the labelledisotype control (negative control). In parallel, identical test batcheswere processed at 4° C. (negative control). FACS analysis was thencarried out using the Guava flow cytometer (Millipore). Kineticevaluation was via measurement of the fluorescence intensity using theguavaSoft 2.6 software (Millipore). A target-mediated specificinternalisation of the anti-CD123 antibodies was observed in variouscancer cell lines (MOLM-13, THP-1, KG-1), whereas the isotype controland the 4° C. batches showed no internalisation.

C-2b Determination of the Co-Localisation of the Internalised CD123Antibody in the Lysosome

The toxic active compound of the antibody drug conjugates is released inthe lysosome, owing to the linker selected. The transport of theantibody to this organelle is therefore crucial. By co-localisation ofthe antibody with marker proteins specific for the lysosome (e.g.surface protein or small GTPasen), it is possible to identify antibodieshaving a suitable profile. To this end, the CD123-positive cells(5×10⁴/well) in 100 μl of medium were initially sown into a 96-MTP(Greiner bio-one, CELLSTAR, 650 180, U-bottom). After addition of theCypHer5E-labelled anti-CD123 antibody (final concentration 20 μg/ml),the batches were incubated in duplicate at 37° C. for 6 h. In each case30 min prior to the end of the incubation time, a lysosome-specific livedye was added to the samples. The lysomes were stained using theCytoPainter LysoGreen indicator reagent (final concentration 1:2000;abeam, ab176826). After incubation, 200 μl of ice-cold FACS buffer(DULBECCO'S PBS, Sigma-Aldrich, No. D8537+3% FBS heat inactivated FBS,Gibco, No. 10500-064) were pipetted into the batch and the cellsuspension was centrifuged at 400×g and 4° C. for 5 min. The pellet wasthen resuspended in 300 μl ice-cold FACS buffer and centrifuged again (4min, 400×g at 4° C.). After the centrifugation, the supernatant wasdiscarded and the pellet was taken up in 30 μl of ice-cold FACS buffer.The samples prepared in this manner were immediately subjected toFACS/image analysis (FlowSight amnis, Millipore). Co-localisation wasevaluated using the specific co-localisation software IDEAS Applicationv6.1. Table 3 summarizes the data from this assay for the anti-CD123antibodies examined.

TABLE 3 Examples Co-localisation [%] TPP-6013 43.5 TPP-5969 26.0TPP-8987 28.0 TPP-8988 41.0 TPP-9476 29.0 7G3 10.0 Isotype control 0.2

Different antibodies showed an improved specific internalization potencyand co-localization compared to the murine antibody.

C-3 In Vitro Tests for Determining Cell Permeability

The cell permeability of a substance can be investigated by means of invitro testing in a flux assay using Caco-2 cells [M. D. Troutman and D.R. Thakker, Pharm. Res. 20 (8), 1210-1224 (2003)]. For this purpose, thecells were cultured for 15-16 days on 24-well filter plates. For thedetermination of permeation, the respective test substance was appliedin a HEPES buffer to the cells either apically (A) or basally (B) andincubated for 2 hours. After 0 hours and after 2 hours, samples weretaken from the cis and trans compartments. The samples were separated byHPLC (Agilent 1200, Böblingen, Germany) using reverse phase columns. TheHPLC system was coupled via a Turbo Ion Spray Interface to a TripleQuadropol mass spectrometer API 4000 (AB SCIEX Deutschland GmbH,Darmstadt, Germany). The permeability was evaluated on the basis of aP_(app) value, which was calculated using the formula published bySchwab et al. [D. Schwab et al., J. Med. Chem. 46, 1716-1725 (2003)]. Asubstance was classified as actively transported when the ratio ofP_(app) (B-A) to P_(app) (A-B) (efflux ratio) was >2 or <0.5. Ofcritical importance for toxophores which are released intracellularly isthe permeability from B to A [P_(app) (B-A)] and the ratio of P_(app)(B-A) to P_(app) (A-B) (efflux ratio): the lower this permeability, theslower the active and passive transport processes of the substancethrough the monolayer of Caco-2 cells. If additionally the efflux ratiodoes not indicate any active transport, the substance may, followingintracellular release, remain longer in the cell. Hence, there is alsomore time available for interaction with the biochemical target (in thiscase: kinesin spindle protein, KSP/Eg5).

Table 4 below sets out permeability data for representative workingexamples from this assay:

TABLE 4 P_(app) (B-A) Working Example [nm/s] Efflux ratio M20 0.89 0.21M21 0.5 1.5

C-4 In Vitro Tests for Determining the Substrate Properties forP-Glycoprotein (P-Gp)

Many tumour cells express transporter proteins for drugs, and thisfrequently accompanies the development of resistance towardscytostatics. Substances which are not substrates of such transporterproteins, such as P-glycoprotein (P-gp) or BCRP, for example, couldtherefore exhibit an improved activity profile.

The substrate properties of a substance for P-gp (ABCB1) were determinedby means of a flux assay using LLC-PK1 cells which overexpress P-gp(L-MDR1 cells) [A. H. Schinkel et al., J. Clin. Invest. 96, 1698-1705(1995)]. For this purpose, the LLC-PK1 cells or L-MDR1 cells werecultured on 96-well filter plates for 3-4 days. For determination of thepermeation, the respective test substance, alone or in the presence ofan inhibitor (such as ivermectin or verapamil, for example), was appliedin a HEPES buffer to the cells either apically (A) or basally (B) andincubated for 2 hours. After 0 hours and after 2 hours, samples weretaken from the cis and trans compartments. The samples were separated byHPLC using reverse phase columns. The HPLC system was coupled via aTurbo Ion Spray Interface to a Triple Quadropol mass spectrometer API3000 (Applied Biosystems Applera, Darmstadt, Germany). The permeabilitywas evaluated on the basis of a P_(app) value, which was calculatedusing the formula published by Schwab et al. [D. Schwab et al., J. Med.Chem. 46, 1716-1725 (2003)]. A substance was classified as P-gpsubstrate when the efflux ratio of P_(app) (B-A) to P_(app) (A-B) was>2.

As further criteria for the evaluation of the P-gp substrate properties,the efflux ratios in L-MDR1 and LLC-PK1 cells or the efflux ratio in thepresence or absence of an inhibitor may be compared. If these valuesdiffer by a factor of more than 2, the substance in question is a P-gpsubstrate.

C-5 Pharmacokinetics

C5a: Identification of the ADC Metabolites after Internalisation InVitro

Description of the method:

Internalisation studies with immunoconjugates are carried out to analysemetabolites formed intracellularly. To this end, human lung tumour cellsNCI H292 (3×10⁵/well) are sown in 6-well plates and incubated overnight(37° C., 5% CO₂). The cells are treated with 10 μg/ml (66 nM) of the ADCto be examined. Internalisation was carried out at 37° C. and 5% CO₂. Atvarious time points (0, 4, 24, 48, 72 h), cell samples are taken forfurther analysis. First, the supernatants (about 5 ml) are harvestedand, after centrifugation (2 min, RT, 1000 rpm Heraeus Variofuge 3.0R),stored at −80° C. The cells are washed with PBS and detached withAccutase, and the cell number is determined. After another washing, adefined number of cells (2×10⁵) is treated with 100 ml of lysis buffer(Mammalian Cell Lysis Kit (Sigma MCL1) and incubated with continuousshaking (Thermomixer, 15 min, 4° C., 650 rpm) in Protein LoBind tubes(Eppendorf Cat. No. 0030 108.116). After the incubation, the lysate iscentrifuged (10 min, 4° C., 12000 g, Eppendorf 5415R) and thesupernatant is harvested. The supernatant obtained is stored at −80° C.All samples are then analysed as follows.

Measurement of the compounds in the culture supernatant or cell lysateis carried out after precipitation of the proteins with methanol oracetonitrile by high-pressure liquid chromatography (HPLC) coupled to atriple-quadrupole mass spectrometer (MS).

For work-up of 50 μl of culture supernatant/cell lysate, 150 μl ofprecipitation reagent (generally acetonitrile) are added and the mixtureis shaken for 10 seconds. The precipitation reagent contains an internalstandard (ISTD) in a suitable concentration (generally in the range of20-100 ng/ml). After 3 minutes of centrifugation at 16000 g, thesupernatant is transferred into an autosampler vial, made up with 500 μlof a buffer suitable for the mobile phase and shaken again.

The two matrix samples are then measured using the HPLC-coupledtriple-quadrupol mass spectrometer API6500 from AB SCIEX DeutschlandGmbH.

For calibration, concentrations of 0.5-2000 μg/l are added to plasmasamples. The detection limit (LOQ) is about 2 μg/l. The linear rangeextends from 2 to 1000 μg/l.

For calibration of the tumour samples, concentrations of 0.5-200 μg/lare added to the supernatant of untreated tumours. The detection limitis 4 μg/l. The linear range extends from 4 to 200 μg/l.

Quality controls for testing validity contain 5 and 50 μg/l.

C5b: Identification of the ADC Metabolites In Vivo

After i.v. administration of 3-30 mg/kg of different ADCs, the plasmaand tumour concentrations of the ADCs and any metabolites occuring canbe measured, and the pharmacokinetic parameters such as clearance (CL),area under the curve (AUC) and half-times (t_(1/2)) can be calculated.

Analysis for Quantification of any Metabolites Occurring

Measurement of the compounds in plasma and tumour is carried out afterprecipitation of the proteins with methanol or acetonitrile byhigh-pressure liquid chromatography (HPLC) coupled to atriple-quadrupole mass spectrometer (MS).

For work-up of 50 μl of plasma, 250 μl of precipitation reagent(generally acetonitrile) are added and the mixture is shaken for 10seconds. The precipitation reagent contains an internal standard (ISTD)in a suitable concentration (generally in the range of 20-100 ng/ml).After 3 minutes of centrifugation at 16000 g, the supernatant istransferred into an autosampler vial, made up with 500 μl of a buffersuitable for the mobile phase and shaken again.

During the work-up of a tumour, the latter is treated with 3 times theamount of extraction buffer. The extraction buffer contains 50 ml ofTissue Protein Extraction Reagent (Pierce, Rockford, IL), two pellets ofComplete-Protease-Inhibitor-Cocktail (Roche Diagnostics GmbH, Mannheim,Germany) and phenylmethylsulphonyl fluoride (Sigma, St. Louis, MO) in afinal concentration of 1 mM. The sample is homogenized twice for 20minutes in a Tissuelyser II (Qiagen), at maximum stroke number. 50 μl ofthe homogenate are transferred into an autosampler vial and made up with150 μl of methanol including ISTD. After 3 minutes of centrifugation at16000 g, 10 μl of the supernatant are made up with 180 μl of a buffersuitable for the mobile phase and shaken again. The tumour sample isthen ready for measuring.

The two matrix samples are then measured using the HPLC-coupledtriple-quadrupol mass spectrometer AP16500 from AB SCIEX DeutschlandGmbH.

For calibration, concentrations of 0.5-2000 μg/l are added to plasmasamples. The detection limit (LOQ) is about 2 μg/l. The linear rangeextends from 2 to 1000 μg/l.

For calibration of the tumour samples, concentrations of 0.5-200 μg/lare added to the supernatant of untreated tumours. The detection limitis 5 μg/l. The linear range extends from 5 to 200 μg/l.

Quality controls for testing validity contain 5 and 50 μg/l, in plasmaadditionally 500 μg/l.

Analysis for Quantification of the Antibodies Used

The antibody part of the ADCs was determined using a ligand bindingassay (ELISA) as total IgG concentration in plasma samples and tumourlysates. Here, the sandwich ELISA format was used. This ELISA had beenqualified and validated for the determination in plasma and tumoursamples. The ELISA plates were coated with anti-human goat IgG Feantibodies. After incubation with the sample, the plates were washed andincubated with a detector conjugate of simian anti-human IgG(H+L)antibody and horseradish peroxidase (HRP). After a further washing step,the HRP substrate was added to OPD and the colour development wasmonitored via absorption at 490 nm. Standard samples having a known IgGconcentration were fitted using a 4-parameter equation. Within the lower(LLOQ) and upper (ULOQ) quantification limits, the unknownconcentrations were determined by interpolation.

C-6 Efficacy Test In Vivo

The in vivo efficacy of the conjugates of this invention has been testedin tumor Xenograft models. The expert is familiar with methods in theprior art against which the effectiveness of the compounds of theinvention can be tested (see for example WO 2005/081711; Polson et al,Cancer Res 2009 Mar. 15; 69 (6): 2358-64).

For example, rodents (e.g. mice) were implanted with a tumor cell lineexpressing the target molecule of the antibody. Then a conjugate of theinvention, an isotype conjugate, a control antibody or isotonic saline(vehicle) were administered to the implanted animals. After anincubation period of several days, the tumor size was determined inconjugate treated animals vs. control groups (control conjugate and/orvehicle). The conjugate treated animals showed a reduced tumor size.

C-6a. Growth Inhibition of Experimental Tumors in Mice

Human tumor cells expressing the target antigen for the antibody-drugconjugate were inoculated subcutaneously into the flank ofimmunocompromised mice (e.g.: NMRI Nude- or SCID mice). 1-10 millioncells were detached from cell culture, centrifuged and re-suspended withmedium or medium/Matrigel. The cell suspension was injectedsubcutaneously into mice. Treatment (i.p. application at a volume of 5mL/kg; Dosing schedule is shown in table x) started after establishmentof the tumor, approximately at a tumor size of 40 mm².

By default, 8 animals were used per treatment group. In addition to thegroups that received the active substances, a group was treated withvehicle only. During the experiment, the tumor area was measuredregularly with calipers in two dimensions (length/width). The tumor areawas determined by length×width. The comparison of the mean tumor area ofthe treatment group with the control group was expressed as T/C area.

C-6b. Efficacy Of Compounds According to the Invention in Human AMLXenoTraft Models

Treatment with the anti CD123 antibody-drug conjugates resulted in asignificant and long-lasting inhibition of tumor growth. Table 5summarizes in vivo efficacy of representative working examples. Primaryread out is the tumor growth inhibition in treatment vs. vehicle groupas indicated by TIC values (determined from the last measured time pointbefore the vehicle group reached a tumor size of >200 mm² and had to besacrificed due to German animal protection law). As a second read outtable 5 also indicates the time period until the treatment groups showedtumor regrowth and the study was terminated.

Exp 1 shows a strong efficacy and clear superiority of ref 257 ml vs 208ml as measured by tumor growth inhibition in THP-1 AML Xenografl model(TIC 0.46 vs. 0.92)

The efficacy of ref 257 ml has also been evaluated in anadditional AMLXenograft models. Exp. 2. confirmed superiority of ref 257 ml vs 208 mlbased on tumor growth inhibition (TIC 0.25 vs 0.42) and also stronglyreduced time until tumor regrowth in the MOLM 13 AML Xenograft model (34days vs 24 days). The examples 1a, 1b, 1c show strong efficacy in twoAML models (Exp.3 and Exp.4), All examples described show superiorityversus ref208 ml (egla vs 208 ml-TIC 0.32 vs 0,42).

The corresponding isotype-ADC controls with the indicated schedule(Q7dx2) were comparable to vehicle controls.

TABLE 5 Treat- Tumor ment Outgrowth Exam- Xenograft Sched- of ADC Exp.ples Model Dosing ule T/C area vs. vehicle Exp 1 Ref THP-1 5 mg/kg Q7dx20.46 Vehicle Day 21 257m1 (Day 21) ADC Day ongoing Ref THP-1 5 mg/kgQ7dx2 0.92 Vehicle Day 21 208m1 (Day 21) ADC Day 21 Exp 2 Ref MOLM-13 5mg/kg Q7dx2 0.42 Vehicle Day 17 208m1 (Day 17) ADC Day 24 Ref MOLM-13 5mg/kg Q7dx2 0.25 Vehicle Day 17 257m1 (Day 17) ADC Day 31 Exp 3 1aMOLM13 5 mg/kg Q7dx2 0.32 Vehicle Day 14 (day 14) ADC Day 25 1b MOLM13 5mg/kg Q7dx2 0.37 Vehicle Day 14 (day 14) ADC Day 25 1c MOLM13 5 mg/kgQ7dx2 0.33 Vehicle Day 14 (day 14) ADC Day 25 Exp 4 1a MV4-11 5 mg/kgQ7dx2 0.35 Vehicle Day 21 (day 21) ADC Day 32 1b MV4-11 5 mg/kg Q7dx20.25 Vehicle Day 21 (day 21) ADC Day 35 1c MV4-11 5 mg/kg Q7dx2 0.32Vehicle Day 21 (day 21) ADC Day 35

Working Examples of Anti-CD123 Antibodies

All examples were carried out using standard methods known to the personskilled in the art, unless described here in detail. Routine methods ofmolecular biology of the examples that follow can be carried out asdescribed in standard laboratory textbooks such as Sambrook et al.,Molecular Cloning: a Laboratory Manual, 2. Edition; Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., 1989.

Generation and Optimization of Humanized Antd-CD123 Antibodies

The antibodies described herein base on two murine antibody clones 7G3and 12F1 described in literature (EP2426148; WO2013/173820). Shortly,the murine CDRs were grafted into human frameworks as described byO'Brien and Jones, (Chapter 40 Humanizing Antibodies by CDR Grafting,p579 Table 4) and also Hwang et al, (Methods 36 (2005) p35-42). For thedefinition of the CDRs according to Kabat, see Andre C. R. Martin,“Protein sequence and structure analysis of antibody variable domains”in Antibody Engineering (Springer Lab Manuals), Eds.: Duebel, S. andKontermann, R., Springer-Verlag, Heidelberg. After comparison of themurine frame sequences (without CDRs) with human germline sequences, asimilar frequently occuring human frame sequence was selected. Thesequences were named using the IMGT system (Lefranc, M.-P., Giudicelli,V., Ginestoux, C., Jabado-Michaloud, J., Folch, G., Bellahcene, F., Wu,Y., Gemrot, E., Brochet, X., Lane, J., Regnier, L., Ehrenmann, F.,Lefranc, G. and Duroux, P. IMGT®, the international ImMunoGeneTicsinformation system@. Nucl. Acids Res, 37, D1006-D1012 (2009);doi:10.1093/nar/gkn838).

The humanized variants were transiently expressed in HEK293 cells.Supernatants were tested in SPR and FACS for the binding to antigen andcells. To reduce immunogenicity and optimize binding properties, singleor multiple mutations were introduced in CDRs and Frameworks. More than100 variants were tested in order to generate and select the final leadcandidates. The antibody variants described herein carry various pointmutations differing from the human germline sequence which may influencetheir properties.

The first campaign based on the murine antibody clone 12F1(WO2013/173820). The grafting of the CDRs was not trivial and had to betested on several human framework backbones in order succeed. The bestgrafted version was identified to be TPP-8988, in which the light chainCDRs were grafted into the human framework backbone IGKV3-20, the heavychain CDRs were grafted into the human framework backbone IGHV3-66. Incomparison, the grafting of the murine CDRs failed in other cases, e.g.for TPP-8613 which showed reduced FACS binding. In this variant, thelight chain CDRs were grafted into the human framework backbone IGKV4-1,the heavy chain CDRs were grafted into the human framework backboneIGHV3-66. Another example that did not yield sufficient expression as agrafted version is TPP-8617, in which the light chain CDRs were graftedinto the human framework backbone IGKV4-1, the heavy chain CDRs weregrafted into the human framework backbone IGHV5-51.

To further de-immunize TPP-8988, single and recombined non-germlineresidues were exchanged to human germline and 66 variants were testedfor expression and binding. The best recombination variant tested wasTPP-9342. Other variants, as for example TPP-9075, TPP-9078, TPP-9104,TPP-9129 or TPP-9130 showed low expression and/or undesirablemultiphasic behavior in the SPR measurements, although the share similarsequences with TPP-8988/TPP9342.

The second campaign based on the murine antibody clone 7G3 (EP2426148).The humanized version TPP-5969 was generated as described above, basedon human germlines IGKV4-1 and IGHV1-69. It can serve as an alternativeto CSL-362 (TPP-5709), a humanized version generated by CSL (EP2606069).

Unfortunately, TPP-5969 showed an undesirable multiphasic bindingbehaviour in the SPR analysis. To abolish this multiphasic binding andto de-immunize TPP-5969 and, single and recombined non-germline residueswere exchanged to human germline and 53 variants were tested forexpression and binding. Most variants, as for example TPP-8465,TPP-8467, TPP-8468, or TPP-9479 showed low expression and multiphasicbehavior in the SPR measurements as well diminished FACS binding,although the share similar sequences with humanized version TPP-5969and/or the final candidates TPP-8987 or TPP-9476.

Only variants, such as e.g. TPP-8695, containing three mutations in theheavychain (N61A, K65Q and Q67R) compared to TPP-5969 showed singledigit nanomolar EC50 values in FACS binding while still binding in amultiphasic fashion in SPR at low expression. Unexpectedly, theintroduction of a single point mutation in the light chain CDR2 (V29L)in TPP8987 abolished the undesireable multiphasic binding properties.Since the expression rates of TPP-8987 were rather low, different heavychain variants were generated in order to find a recombination ofmutations that allows reasonable expression together withnon-multiphasic SPR binding properties and positive FACS results. Thoseassets are represented by TPP-9476 which only differs from TPP-8987 inthe R67Q backmutation but expresses 10-fold higher while retaining itsfavorable binding features.

Determination of the binding affinity of the antibodies by surfaceplasmon resonance:

Surface plasmon resonance experiments for quantitative binding analysiswere carried out using a Biacore T200 instrument (GE Healthcare Biacore,Inc.). Here the antibodies to be examined were fixed with the aid of ananti-human Fe antibody (“Human Antibody Capture Kit”, BR-1008-39, GEHealthcare Biacore, Inc.) amine-coupled to the sensor chip surface.Amine coupling was performed according to the manufacturer'sinstructions using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimidehydrochloride (EDC), N-hydroxysuccinimide (NHS) and ethanolamine HCl pH8.5 (“Amine Coupling Kit” BR-1000-50, GE Healthcare Biacore, Inc.). Forthe analyses, Series S sensor chips CM5 (GE Healthcare Biacore, Inc.)were used with the mobile buffer HBS-EP+(10 mM HEPES pH 7.4, 150 mMNaCl, 3 mM EDTA, 0.05% Surfactant P20). All experimental steps werecarried out at 25° C. After fixation of the anti-CD123 antibodies to beexamined, injections of the extracellular domain of IL3Rα (analyte, R&DSystems) at concentrations of 100 nM were carried out, and after eachantigen injection the sensor surface was regenerated with glycine HCl pH2.0. Prior to another analyte injection, antibodies were in each casefixed as above, under identical conditions. For all measurements, anupstream flow cell only containing immobilized amine-coupled anti-humanFe antibody was used as reference cell. Evaluation of the sensorgramsobtained was, after double referencing (subtraction of the referenceflow cell signal and a buffer injection), carried out by a global fitbased on a 1:1 Langmuir binding model with the aid of the Biacore T200evaluation software (GE Healthcare Biacore, Inc.).

TABLE 6a Recombinant antigen (IL3R3alpha/CD123) used for affinitydetermination Nomenclature Description Origin Cat. No. (R&D) TPP-5521IL3Ra aa20-305 murine 203-IL

TABLE 6b Monovalent K_(D) values of the anti-CD123 antibodies determinedwith Biacore using IL3Ra protein (TPP-5521 as analyte) Chi2 % ofMultiphasic TPP K_(D) [nM] Rmax behaviour TPP-5969 Kinetic evaluation —Yes not applicable TPP-8987 1.24 No TPP-9476 7.2 No TPP-6013 0.8 1 NoTPP-8988 0.33 No TPP-9342

Binding of Anti-CD123 Antibodies to Various Antigen-Expressing CancerCell Lines

Binding of the anti-CD123 antibodies was examined by flow cytometryusing human haematological cell line MOLM-13. To this end, the cells(5×10⁵ cells/well) were incubated in FACS buffer (PBS without Ca/Mg, 3%FCS, Biochrom) with 10 μg/ml primary antibody solution (startconcentration) on ice for 30-45 min protected from light. A doseactivity curve (1:5 dilution) was plotted. After the incubation, 200 μlof ice-cold FACS buffer were added using a pipette, and the cellsuspension was centrifuged at 4° C. 400 g for 4 min. The cell pellet waswashed with 300 μl of ice-cold FACS buffer and the pellet obtained wasthen resuspended in 100 μl FACS buffer and incubated again withsecondary antibody (monoclonal anti-kappa light chains-FITC antibody,Sigma, No. SAB4700605) in a 1:10 dilution on ice for 30 min. The cellswere then washed with ice-cold FACS buffer and adjusted to a cellconcentration of 0.5×10⁶ cell/ml prior to flow cytometry using a Guavaflow cytometer (Millipore). Propidium iodide (final concentration 1μg/ml) was used for live staining. The results were shown as EC₅₀ valuesdetermined based on the dose response curve and efficacy compared tomaximal binding. Surprisingly a significant improved binding behaviourcould be detected with TPP-8988 compared to TPP-5969 and TPP-8988compared to TPP-6013 (Table 7A) Most humanized variants of TPP-5969showed no or insufficient binding to the target (7B) as well as thehumanized variants of TPP-6013 (7C; see generation and optimization).

TABLE 7 FACS analysis: Binding of the anti-CD123 antibodies to MOLM-13cancer cell line. A improved humanized anti-CD123 antibodies; B TPP-5963variants; C TPP-6013 variants MOLM-13 Efficacy TPP EC50 [nM] [%] ATPP-5969 5.3 34 TPP-8987 3.2 100 TPP-9476 2.8 100 TPP-6013 2.0 100TPP-8988 1.9 100 TPP-9342 B TPP-8465 No binding TPP-8467 No bindingTPP-8468 No binding TPP-8476 No binding TPP-8695  1.12 36 C TPP-8613 3.0100 TPP-8617 1.4 100 TPP-9075  5.37 100 TPP-9078 No binding — TPP-910425.3  83 TPP-9129 No binding — TPP-9130  3.01 100

TABLE List of Sequenzes Sequence Sequence TPP ID Sequence Name RegionType SEQ ID TPP-5709 CSL362- VH PRT SEQ ID hIgG1Kappa NO: 1 TPP-5709CSL362- HCDR1 PRT SEQ ID hIgG1Kappa NO: 2 TPP-5709 CSL362- HCDR2 PRT SEQID hIgG1Kappa NO: 3 TPP-5709 CSL362- HCDR3 PRT SEQ ID hIgG1Kappa NO: 4TPP-5709 CSL362- VL PRT SEQ ID hIgG1Kappa NO: 5 TPP-5709 CSL362- LCDR1PRT SEQ ID hIgG1Kappa NO: 6 TPP-5709 CSL362- LCDR2 PRT SEQ ID hIgG1KappaNO: 7 TPP-5709 CSL362- LCDR3 PRT SEQ ID hIgG1Kappa NO: 8 TPP-5709CSL362- Heavy PRT SEQ ID hIgG1Kappa Chain NO: 9 TPP-5709 CSL362- LightPRT SEQ ID hIgG1Kappa Chain NO: 10 TPP-5968 HU7G3-1- VH PRT SEQ IDhIgG1Kappa NO: 11 TPP-5968 HU7G3-1- HCDR1 PRT SEQ ID hIgG1Kappa NO: 12TPP-5968 HU7G3-1- HCDR2 PRT SEQ ID hIgG1Kappa NO: 13 TPP-5968 HU7G3-1-HCDR3 PRT SEQ ID hIgG1Kappa NO: 14 TPP-5968 HU7G3-1- VL PRT SEQ IDhIgG1Kappa NO: 15 TPP-5968 HU7G3-1- LCDR1 PRT SEQ ID hIgG1Kappa NO: 16TPP-5968 HU7G3-1- LCDR2 PRT SEQ ID hIgG1Kappa NO: 17 TPP-5968 HU7G3-1-LCDR3 PRT SEQ ID hIgG1Kappa NO: 18 TPP-5968 HU7G3-1-  Heavy PRT SEQ IDhIgG1Kappa Chain NO: 19 TPP-5968 HU7G3-1-  Light PRT SEQ ID hIgG1KappaChain NO: 20 TPP-5969 HU7G3-4- VH PRT SEQ ID hIgG1Kappa NO: 21 TPP-5969HU7G3-4- HCDR1 PRT SEQ ID hIgG1Kappa NO: 22 TPP-5969 HU7G3-4- HCDR2 PRTSEQ ID hIgG1Kappa NO: 23 TPP-5969 HU7G3-4- HCDR3 PRT SEQ ID hIgG1KappaNO: 24 TPP-5969 HU7G3-4- VL PRT SEQ ID hIgG1Kappa NO: 25 TPP-5969HU7G3-4- LCDR1 PRT SEQ ID hIgG1Kappa NO: 26 TPP-5969 HU7G3-4- LCDR2 PRTSEQ ID hIgG1Kappa NO: 27 TPP-5969 HU7G3-4- LCDR3 PRT SEQ ID hIgG1KappaNO: 28 TPP-5969 HU7G3-4- Heavy PRT SEQ ID hIgG1Kappa Chain NO: 29TPP-5969 HU7G3-4- Light PRT SEQ ID hIgG1Kappa Chain NO: 30 TPP-5971HU7G3-3- VH PRT SEQ ID hIgG1Kappa NO: 31 TPP-5971 HU7G3-3- HCDR1 PRT SEQID hIgG1Kappa NO: 32 TPP-5971 HU7G3-3- HCDR2 PRT SEQ ID hIgG1Kappa NO:33 TPP-5971 HU7G3-3- HCDR3 PRT SEQ ID hIgG1Kappa NO: 34 TPP-5971HU7G3-3- VL PRT SEQ ID hIgG1Kappa NO: 35 TPP-5971 HU7G3-3- LCDR1 PRT SEQID hIgG1Kappa NO: 36 TPP-5971 HU7G3-3- LCDR2 PRT SEQ ID hIgG1Kappa NO:37 TPP-5971 HU7G3-3- LCDR3 PRT SEQ ID hIgG1Kappa NO: 38 TPP-5971HU7G3-3- Heavy PRT SEQ ID hIgG1Kappa Chain NO: 39 TPP-5971 HU7G3-3-Light PRT SEQ ID hIgG1Kappa Chain NO: 40 TPP-6013 antiCD123-12F1- VH PRTSEQ ID hIgG1Kappa NO: 41 TPP-6013 antiCD123-12F1- HCDR1 PRT SEQ IDhIgG1Kappa NO: 42 TPP-6013 antiCD123-12F1- HCDR2 PRT SEQ ID hIgG1KappaNO: 43 TPP-6013 antiCD123-12F1- HCDR3 PRT SEQ ID hIgG1Kappa NO: 44TPP-6013 antiCD123-12F1- VL PRT SEQ ID hIgG1Kappa NO: 45 TPP-6013antiCD123-12F1- LCDR1 PRT SEQ ID hIgG1Kappa NO: 46 TPP-6013antiCD123-12F1- LCDR2 PRT SEQ ID hIgG1Kappa NO: 47 TPP-6013antiCD123-12F1- LCDR3 PRT SEQ ID hIgG1Kappa NO: 48 TPP-6013antiCD123-12F1- Heavy PRT SEQ ID hIgG1Kappa Chain NO: 49 TPP-6013antiCD123-12F1- Light PRT SEQ ID hIgG1Kappa Chain NO: 50 TPP-8465GL-TPP5969- VH PRT SEQ ID hIgG1kappa-gl05 NO: 51 TPP-8465 GL-TPP5969-HCDR1 PRT SEQ ID hIgG1kappa-gl05 NO: 52 TPP-8465 GL-TPP5969- HCDR2 PRTSEQ ID hIgG1kappa-gl05 NO: 53 TPP-8465 GL-TPP5969- HCDR3 PRT SEQ IDhIgG1kappa-gl05 NO: 54 TPP-8465 GL-TPP5969- VL PRT SEQ IDhIgG1kappa-gl05 NO: 55 TPP-8465 GL-TPP5969- LCDR1 PRT SEQ IDhIgG1kappa-gl05 NO: 56 TPP-8465 GL-TPP5969- LCDR2 PRT SEQ IDhIgG1kappa-gl05 NO: 57 TPP-8465 GL-TPP5969- LCDR3 PRT SEQ IDhIgG1kappa-gl05 NO: 58 TPP-8465 GL-TPP5969- Heavy PRT SEQ IDhIgG1kappa-gl05 Chain NO: 59 TPP-8465 GL-TPP5969- Light PRT SEQ IDhIgG1kappa-gl05 Chain NO: 60 TPP-8467 GL-TPP5969- VH PRT SEQ IDhIgG1kappa-gl07 NO: 61 TPP-8467 GL-TPP5969- HCDR1 PRT SEQ IDhIgG1kappa-gl07 NO: 62 TPP-8467 GL-TPP5969- HCDR2 PRT SEQ IDhIgG1kappa-gl07 NO: 63 TPP-8467 GL-TPP5969- HCDR3 PRT SEQ IDhIgG1kappa-gl07 NO: 64 TPP-8467 GL-TPP5969- VL PRT SEQ IDhIgG1kappa-gl07 NO: 65 TPP-8467 GL-TPP5969- LCDR1 PRT SEQ IDhIgG1kappa-gl07 NO: 66 TPP-8467 GL-TPP5969- LCDR2 PRT SEQ IDhIgG1kappa-gl07 NO: 67 TPP-8467 GL-TPP5969- LCDR3 PRT SEQ IDhIgG1kappa-gl07 NO: 68 TPP-8467 GL-TPP5969- Heavy PRT SEQ IDhIgG1kappa-gl07 Chain NO: 69 TPP-8467 GL-TPP5969- Light PRT SEQ IDhIgG1kappa-gl07 Chain NO: 70 TPP-8468 GL-TPP5969- VH PRT SEQ IDhIgG1kappa-gl08 NO: 71 TPP-8468 GL-TPP5969- HCDR1 PRT SEQ IDhIgG1kappa-gl08 NO: 72 TPP-8468 GL-TPP5969- HCDR2 PRT SEQ IDhIgG1kappa-gl08 NO: 73 TPP-8468 GL-TPP5969- HCDR3 PRT SEQ IDhIgG1kappa-gl08 NO: 74 TPP-8468 GL-TPP5969- VL PRT SEQ IDhIgG1kappa-gl08 NO: 75 TPP-8468 GL-TPP5969- LCDR1 PRT SEQ IDhIgG1kappa-gl08 NO: 76 TPP-8468 GL-TPP5969- LCDR2 PRT SEQ IDhIgG1kappa-gl08 NO: 77 TPP-8468 GL-TPP5969- LCDR3 PRT SEQ IDhIgG1kappa-gl08 NO: 78 TPP-8468 GL-TPP5969- Heavy PRT SEQ IDhIgG1kappa-gl08 Chain NO: 79 TPP-8468 GL-TPP5969- Light PRT SEQ IDhIgG1kappa-gl08 Chain NO: 80 TPP-8476 GL-TPP5969- VH PRT SEQ IDhIgG1kappa-gl16 NO: 81 TPP-8476 GL-TPP5969- HCDR1 PRT SEQ IDhIgG1kappa-gl16 NO: 82 TPP-8476 GL-TPP5969- HCDR2 PRT SEQ IDhIgG1kappa-gl16 NO: 83 TPP-8476 GL-TPP5969- HCDR3 PRT SEQ IDhIgG1kappa-gl16 NO: 84 TPP-8476 GL-TPP5969- VL PRT SEQ IDhIgG1kappa-gl16 NO: 85 TPP-8476 GL-TPP5969- LCDR1 PRT SEQ IDhIgG1kappa-gl16 NO: 86 TPP-8476 GL-TPP5969- LCDR2 PRT SEQ IDhIgG1kappa-gl16 NO: 87 TPP-8476 GL-TPP5969- LCDR3 PRT SEQ IDhIgG1kappa-gl16 NO: 88 TPP-8476 GL-TPP5969- Heavy PRT SEQ IDhIgG1kappa-gl16 Chain NO: 89 TPP-8476 GL-TPP5969- Light PRT SEQ IDhIgG1kappa-gl16 Chain NO: 90 TPP-8613 Hum-TPP6013-13 VH PRT SEQ ID NO:91 TPP-8613 Hum-TPP6013-13 HCDR1 PRT SEQ ID NO: 92 TPP-8613Hum-TPP6013-13 HCDR2 PRT SEQ ID NO: 93 TPP-8613 Hum-TPP6013-13 HCDR3 PRTSEQ ID NO: 94 TPP-8613 Hum-TPP6013-13 VL PRT SEQ ID NO: 95 TPP-8613Hum-TPP6013-13 LCDR1 PRT SEQ ID NO: 96 TPP-8613 Hum-TPP6013-13 LCDR2 PRTSEQ ID NO: 97 TPP-8613 Hum-TPP6013-13 LCDR3 PRT SEQ ID NO: 98 TPP-8613Hum-TPP6013-13 Heavy PRT SEQ ID Chain NO: 99 TPP-8613 Hum-TPP6013-13Light PRT SEQ ID Chain NO: 100 TPP-8617 Hum-TPP6013-17 VH PRT SEQ ID NO:101 TPP-8617 Hum-TPP6013-17 HCDR1 PRT SEQ ID NO: 102 TPP-8617Hum-TPP6013-17 HCDR2 PRT SEQ ID NO: 103 TPP-8617 Hum-TPP6013-17 HCDR3PRT SEQ ID NO: 104 TPP-8617 Hum-TPP6013-17 VL PRT SEQ ID NO: 105TPP-8617 Hum-TPP6013-17 LCDR1 PRT SEQ ID NO: 106 TPP-8617 Hum-TPP6013-17LCDR2 PRT SEQ ID NO: 107 TPP-8617 Hum-TPP6013-17 LCDR3 PRT SEQ ID NO:108 TPP-8617 Hum-TPP6013-17 Heavy PRT SEQ ID Chain NO: 109 TPP-8617Hum-TPP6013-17 Light PRT SEQ ID Chain NO: 110 TPP-8695 Variant4- VH PRTSEQ ID hIgG1kappa NO: 111 TPP-8695 Variant4- HCDR1 PRT SEQ ID hIgG1kappaNO: 112 TPP-8695 Variant4- HCDR2 PRT SEQ ID hIgG1kappa NO: 113 TPP-8695Variant4- HCDR3 PRT SEQ ID hIgG1kappa NO: 114 TPP-8695 Variant4- VL PRTSEQ ID hIgG1kappa NO: 115 TPP-8695 Variant4- LCDR1 PRT SEQ ID hIgG1kappaNO: 116 TPP-8695 Variant4- LCDR2 PRT SEQ ID hIgG1kappa NO: 117 TPP-8695Variant4- LCDR3 PRT SEQ ID hIgG1kappa NO: 118 TPP-8695 Variant4- HeavyPRT SEQ ID hIgG1kappa Chain NO: 119 TPP-8695 Variant4- Light PRT SEQ IDhIgG1kappa Chain NO: 120 TPP-8987 Hum 5969 B10- VH PRT SEQ ID hIgG1KappaNO: 121 TPP-8987 Hum 5969 B10- HCDR1 PRT SEQ ID hIgG1Kappa NO: 122TPP-8987 Hum 5969 B10- HCDR2 PRT SEQ ID hIgG1Kappa NO: 123 TPP-8987 Hum5969 B10- HCDR3 PRT SEQ ID hIgG1Kappa NO: 124 TPP-8987 Hum 5969 B10- VLPRT SEQ ID hIgG1Kappa NO: 125 TPP-8987 Hum 5969 B10- LCDR1 PRT SEQ IDhIgG1Kappa NO: 126 TPP-8987 Hum 5969 B10- LCDR2 PRT SEQ ID hIgG1KappaNO: 127 TPP-8987 Hum 5969 B10- LCDR3 PRT SEQ ID hIgG1Kappa NO: 128TPP-8987 Hum 5969 B10- Heavy PRT SEQ ID hIgG1Kappa Chain NO: 129TPP-8987 Hum 5969 B10- Light PRT SEQ ID hIgG1Kappa Chain NO: 130TPP-8988 Hum 6013 B2- VH PRT SEQ ID hIgG1Kappa NO: 131 TPP-8988 Hum 6013B2- HCDR1 PRT SEQ ID hIgG1Kappa NO: 132 TPP-8988 Hum 6013 B2- HCDR2 PRTSEQ ID hIgG1Kappa NO: 133 TPP-8988 Hum 6013 B2- HCDR3 PRT SEQ IDhIgG1Kappa NO: 134 TPP-8988 Hum 6013 B2- VL PRT SEQ ID hIgG1Kappa NO:135 TPP-8988 Hum 6013 B2- LCDR1 PRT SEQ ID hIgG1Kappa NO: 136 TPP-8988Hum 6013 B2- LCDR2 PRT SEQ ID hIgG1Kappa NO: 137 TPP-8988 Hum 6013 B2-LCDR3 PRT SEQ ID hIgG1Kappa NO: 138 TPP-8988 Hum 6013 B2- Heavy PRT SEQID hIgG1Kappa Chain NO: 139 TPP-8988 Hum 6013 B2- Light PRT SEQ IDhIgG1Kappa Chain NO: 140 TPP-9075 GL-8988- VH PRT SEQ ID hIgGkappa-gl1NO: 141 TPP-9075 GL-8988- HCDR1 PRT SEQ ID hIgGkappa-gl1 NO: 142TPP-9075 GL-8988- HCDR2 PRT SEQ ID hIgGkappa-gl1 NO: 143 TPP-9075GL-8988- HCDR3 PRT SEQ ID hIgGkappa-gl1 NO: 144 TPP-9075 GL-8988- VL PRTSEQ ID hIgGkappa-gl1 NO: 145 TPP-9075 GL-8988- LCDR1 PRT SEQ IDhIgGkappa-gl1 NO: 146 TPP-9075 GL-8988- LCDR2 PRT SEQ ID hIgGkappa-gl1NO: 147 TPP-9075 GL-8988- LCDR3 PRT SEQ ID hIgGkappa-gl1 NO: 148TPP-9075 GL-8988- Heavy PRT SEQ ID hIgGkappa-gl1 Chain NO: 149 TPP-9075GL-8988- Light PRT SEQ ID hIgGkappa-gl1 Chain NO: 150 TPP-9078 GL-8988-VH PRT SEQ ID hIgGkappa-gl4 NO: 151 TPP-9078 GL-8988- HCDR1 PRT SEQ IDhIgGkappa-gl4 NO: 152 TPP-9078 GL-8988- HCDR2 PRT SEQ ID hIgGkappa-gl4NO: 153 TPP-9078 GL-8988- HCDR3 PRT SEQ ID hIgGkappa-gl4 NO: 154TPP-9078 GL-8988- VL PRT SEQ ID hIgGkappa-gl4 NO: 155 TPP-9078 GL-8988-LCDR1 PRT SEQ ID hIgGkappa-gl4 NO: 156 TPP-9078 GL-8988- LCDR2 PRT SEQID hIgGkappa-gl4 NO: 157 TPP-9078 GL-8988- LCDR3 PRT SEQ IDhIgGkappa-gl4 NO: 158 TPP-9078 GL-8988- Heavy PRT SEQ ID hIgGkappa-gl4Chain NO: 159 TPP-9078 GL-8988- Light PRT SEQ ID hIgGkappa-gl4 Chain NO:160 TPP-9104 GL-8988- VH PRT SEQ ID hIgGkappa-gl56 NO: 161 TPP-9104GL-8988- HCDR1 PRT SEQ ID hIgGkappa-gl56 NO: 162 TPP-9104 GL-8988- HCDR2PRT SEQ ID hIgGkappa-gl56 NO: 163 TPP-9104 GL-8988- HCDR3 PRT SEQ IDhIgGkappa-gl56 NO: 164 TPP-9104 GL-8988- VL PRT SEQ ID hIgGkappa-gl56NO: 165 TPP-9104 GL-8988- LCDR1 PRT SEQ ID hIgGkappa-gl56 NO: 166TPP-9104 GL-8988- LCDR2 PRT SEQ ID hIgGkappa-gl56 NO: 167 TPP-9104GL-8988- LCDR3 PRT SEQ ID hIgGkappa-gl56 NO: 168 TPP-9104 GL-8988- HeavyPRT SEQ ID hIgGkappa-gl56 Chain NO: 169 TPP-9104 GL-8988- Light PRT SEQID hIgGkappa-gl56 Chain NO: 170 TPP-9129 GL-8988- VH PRT SEQ IDhIgGkappa-gl46 NO: 171 TPP-9129 GL-8988- HCDR1 PRT SEQ ID hIgGkappa-gl46NO: 172 TPP-9129 GL-8988- HCDR2 PRT SEQ ID hIgGkappa-gl46 NO: 173TPP-9129 GL-8988- HCDR3 PRT SEQ ID hIgGkappa-gl46 NO: 174 TPP-9129GL-8988- VL PRT SEQ ID hIgGkappa-gl46 NO: 175 TPP-9129 GL-8988- LCDR1PRT SEQ ID hIgGkappa-gl46 NO: 176 TPP-9129 GL-8988- LCDR2 PRT SEQ IDhIgGkappa-gl46 NO: 177 TPP-9129 GL-8988- LCDR3 PRT SEQ ID hIgGkappa-gl46NO: 178 TPP-9129 GL-8988- Heavy PRT SEQ ID hIgGkappa-gl46 Chain NO: 179TPP-9129 GL-8988- Light PRT SEQ ID hIgGkappa-gl46 Chain NO: 180 TPP-9130GL-8988- VH PRT SEQ ID hIgGkappa-gl47 NO: 181 TPP-9130 GL-8988- HCDR1PRT SEQ ID hIgGkappa-gl47 NO: 182 TPP-9130 GL-8988- HCDR2 PRT SEQ IDhIgGkappa-gl47 NO: 183 TPP-9130 GL-8988- HCDR3 PRT SEQ ID hIgGkappa-gl47NO: 184 TPP-9130 GL-8988- VL PRT SEQ ID hIgGkappa-gl47 NO: 185 TPP-9130GL-8988- LCDR1 PRT SEQ ID hIgGkappa-gl47 NO: 186 TPP-9130 GL-8988- LCDR2PRT SEQ ID hIgGkappa-gl47 NO: 187 TPP-9130 GL-8988- LCDR3 PRT SEQ IDhIgGkappa-gl47 NO: 188 TPP-9130 GL-8988- Heavy PRT SEQ ID hIgGkappa-gl47Chain NO: 189 TPP-9130 GL-8988- Light PRT SEQ ID hIgGkappa-gl47 ChainNO: 190 TPP-9342 8988-gl58- VH PRT SEQ ID hIgG1kappa NO: 191 TPP-93428988-gl58- HCDR1 PRT SEQ ID hIgG1kappa NO: 192 TPP-9342 8988-gl58- HCDR2PRT SEQ ID hIgG1kappa NO: 193 TPP-9342 8988-gl58- HCDR3 PRT SEQ IDhIgG1kappa NO: 194 TPP-9342 8988-gl58- VL PRT SEQ ID hIgG1kappa NO: 195TPP-9342 8988-gl58- LCDR1 PRT SEQ ID hIgG1kappa NO: 196 TPP-93428988-gl58- LCDR2 PRT SEQ ID hIgG1kappa NO: 197 TPP-9342 8988-gl58- LCDR3PRT SEQ ID hIgG1kappa NO: 198 TPP-9342 8988-gl58- Heavy PRT SEQ IDhIgG1kappa Chain NO: 199 TPP-9342 8988-gl58- Light PRT SEQ ID hIgG1kappaChain NO: 200 TPP-9476 5969-gl44- VH PRT SEQ ID hIgG1kappa NO: 201TPP-9476 5969-gl44- HCDR1 PRT SEQ ID hIgG1kappa NO: 202 TPP-94765969-gl44- HCDR2 PRT SEQ ID hIgG1kappa NO: 203 TPP-9476 5969-gl44- HCDR3PRT SEQ ID hIgG1kappa NO: 204 TPP-9476 5969-gl44- VL PRT SEQ IDhIgG1kappa NO: 205 TPP-9476 5969-gl44- LCDR1 PRT SEQ ID hIgG1kappa NO:206 TPP-9476 5969-gl44- LCDR2 PRT SEQ ID hIgG1kappa NO: 207 TPP-94765969-gl44- LCDR3 PRT SEQ ID hIgG1kappa NO: 208 TPP-9476 5969-gl44- HeavyPRT SEQ ID hIgG1kappa Chain NO: 209 TPP-9476 5969-gl44- Light PRT SEQ IDhIgG1kappa Chain NO: 210 TPP-9479 5969-gl47- VH PRT SEQ ID hIgG1kappaNO: 211 TPP-9479 5969-gl47- HCDR1 PRT SEQ ID hIgG1kappa NO: 212 TPP-94795969-gl47- HCDR2 PRT SEQ ID hIgG1kappa NO: 213 TPP-9479 5969-gl47- HCDR3PRT SEQ ID hIgG1kappa NO: 214 TPP-9479 5969-gl47- VL PRT SEQ IDhIgG1kappa NO: 215 TPP-9479 5969-gl47- LCDR1 PRT SEQ ID hIgG1kappa NO:216 TPP-9479 5969-gl47- LCDR2 PRT SEQ ID hIgG1kappa NO: 217 TPP-94795969-gl47- LCDR3 PRT SEQ ID hIgG1kappa NO: 218 TPP-9479 5969-gl47- HeavyPRT SEQ ID hIgG1kappa Chain NO: 219 TPP-9479 5969-gl47- Light PRT SEQ IDhIgG1kappa Chain NO: 220

What is claimed is: 1-20. (canceled)
 21. An antibody or antigen-bindingantibody fragment that binds to CD123, comprising a variable heavy chaincomprising the variable CDR1 sequence of the heavy chain, as shown inSEQ ID NO: 132, the variable CDR2 sequence of the heavy chain, as shownin SEQ ID NO: 133, and the variable CDR3 sequence of the heavy chain, asshown in SEQ ID NO: 134; and a variable light chain comprising thevariable CDR1 sequence of the light chain, as shown in SEQ ID NO: 136,the variable CDR2 sequence of the light chain, as shown in SEQ ID NO:137, and the variable CDR3 sequence of the light chain, as shown in SEQID NO:
 138. 22. The antibody or antigen-binding antibody fragment ofclaim 21, wherein the variable heavy chain comprises SEQ ID NO:
 131. 23.The antibody or antigen-binding antibody fragment of claim 21, whereinthe variable light chain comprises SEQ ID NO:
 135. 24. The antibody orantigen-binding antibody fragment of claim 22, wherein the variablelight chain comprises SEQ ID NO:
 135. 25. The antibody orantigen-binding antibody fragment of claim 21, comprising a heavy chaincomprising SEQ ID NO:
 139. 26. The antibody or antigen-binding antibodyfragment of claim 21, comprising a light chain comprising SEQ ID NO:140.
 27. The antibody or antigen-binding antibody fragment of claim 25,comprising a light chain comprising SEQ ID NO:
 140. 28. A pharmaceuticalcomposition comprising the antibody or antigen-binding antibody fragmentof claim 21, and an inert non-toxic pharmaceutically suitable auxiliary.29. A method for treatment of a disease associated with CD123expression, comprising administering to a patient in need thereof aneffective amount of the antibody or antigen-binding antibody fragment ofclaim
 21. 30. A pharmaceutical composition comprising the antibody orantigen-binding antibody fragment of claim 24, and an inert non-toxicpharmaceutically suitable auxiliary.
 31. A method for treatment of adisease associated with CD123 expression, comprising administering to apatient in need thereof an effective amount of the antibody orantigen-binding antibody fragment of claim
 24. 32. A pharmaceuticalcomposition comprising the antibody or antigen-binding antibody fragmentof claim 27, and an inert non-toxic pharmaceutically suitable auxiliary.33. A method for treatment of a disease associated with CD123expression, comprising administering to a patient in need thereof aneffective amount of the antibody or antigen-binding antibody fragment ofclaim 27.